HIV-1 broadly neutralizing antibodies

ABSTRACT

The present application relates broadly neutralizing monoclonal antibodies directed against HIV-1. In particular, monoclonal antibodies VRC-PG04 and VRC-PG05 are disclosed.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation-in-part application of internationalpatent application Serial No. PCT/US2011/052933 filed Sep. 23, 2011,which published as PCT Publication No. WO 2012/040562 on Mar. 29, 2012,which claims priority to U.S. provisional patent application Ser. No.61/386,211 filed Sep. 24, 2010 and U.S. provisional patent applicationSer. No. 61/515,528 filed Aug. 5, 2011.

The foregoing application, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 12, 2012, isnamed 49094992.txt and is 40,911 bytes in size.

FIELD OF THE INVENTION

This application relates to human neutralizing monoclonal antibodiesspecific for HIV-1, such as broad and potent neutralizing monoclonalantibodies specific for HIV-1 and their manufacture and use. Broadneutralization suggests that the antibodies can neutralize HIV-1isolates from different individuals. Such antibodies are useful inpharmaceutical compositions for the prevention and treatment of HIV, andfor the diagnosis and monitoring of HIV infection and for design of HIVvaccine immunogens.

BACKGROUND OF THE INVENTION

AIDS, or Acquired Immunodeficiency Syndrome, is caused by humanimmunodeficiency virus (HIV) and is characterized by several clinicalfeatures including wasting syndromes, central nervous systemdegeneration and profound immunosuppression that results inopportunistic infections and malignancies. HIV is a member of thelentivirus family of animal retroviruses, which include the visna virusof sheep and the bovine, feline, and simian immunodeficiency viruses(SIV). Two closely related types of HIV, designated HIV-1 and HIV-2,have been identified thus far, of which HIV-1 is by far the most commoncause of AIDS. However, HIV-2, which differs in genomic structure andantigenicity, causes a similar clinical syndrome.

An infectious HIV particle consists of two identical strands of RNA,each approximately 9.2 kb long, packaged within a core of viralproteins. This core structure is surrounded by a phospholipid bilayerenvelope derived from the host cell membrane that also includesvirallyencoded membrane proteins (Abbas et al., Cellular and MolecularImmunology, 4th edition, W.B. Saunders Company, 2000, p. 454). The HIVgenome has the characteristic 5′-LTR-Gag-Pol-Env-LTR-3′ organization ofthe retrovirus family. Long terminal repeats (LTRs) at each end of theviral genome serve as binding sites for transcriptional regulatoryproteins from the host and regulate viral integration into the hostgenome, viral gene expression, and viral replication.

The HIV genome encodes several structural proteins. The gag gene encodesstructural proteins of the nucleocapsid core and matrix. The pol geneencodes reverse transcriptase (RT), integrase (IN), and viral protease(PR) enzymes required for viral replication. The tat gene encodes aprotein that is required for elongation of viral transcripts. The revgene encodes a protein that promotes the nuclear export of incompletelyspliced or unspliced viral RNAs. The vif gene product enhances theinfectivity of viral particles. The vpr gene product promotes thenuclear import of viral DNA and regulates G2 cell cycle arrest. The vpuand nef genes encode proteins that down regulate host cell CD4expression and enhance release of virus from infected cells. The envgene encodes the viral envelope glycoprotein that is translated as a160-kilodalton (kDa) precursor (gp160) and cleaved by a cellularprotease to yield the external 120-kDa envelope glycoprotein (gp120) andthe transmembrane 41-kDa envelope glycoprotein (gp41), which arerequired for the infection of cells (Abbas et al., Cellular andMolecular Immunology, 4th edition, W.B. Saunders Company, 2000, pp.454-456). gp140 is a modified form of the Env glycoprotein, whichcontains the external 120-kDa envelope glycoprotein portion and theextracellular part of the gp41 portion of Env and has characteristics ofboth gp120 and gp41. The nef gene is conserved among primatelentiviruses and is one of the first viral genes that is transcribedfollowing infection. In vitro, several functions have been described,including down-regulation of CD4 and MHC class I surface expression,altered T-cell signaling and activation, and enhanced viral infectivity.

HIV infection initiates with gp120 on the viral particle binding to theCD4 and chemokine receptor molecules (e.g., CXCR4, CCR5) on the cellmembrane of target cells such as CD4+ T-cells, macrophages and dendriticcells. The bound virus fuses with the target cell and reversetranscribes the RNA genome. The resulting viral DNA integrates into thecellular genome, where it directs the production of new viral RNA, andthereby viral proteins and new virions. These virions bud from theinfected cell membrane and establish productive infections in othercells. This process also kills the originally infected cell. HIV canalso kill cells indirectly because the CD4 receptor on uninfectedT-cells has a strong affinity for gp120 expressed on the surface ofinfected cells. In this case, the uninfected cells bind, via the CD4receptor-gp120 interaction, to infected cells and fuse to form asyncytium, which cannot survive. Destruction of CD4+ T-lymphocytes,which are critical to immune defense, is a major cause of theprogressive immune dysfunction that is the hallmark of AIDS diseaseprogression. The loss of CD4+ T cells seriously impairs the body'sability to fight most invaders, but it has a particularly severe impacton the defenses against viruses, fungi, parasites and certain bacteria,including mycobacteria.

Research on the Env glycoprotein has shown that the virus has manyeffective protective mechanisms with few vulnerabilities (Wyatt &Sodroski, Science. 1998 Jun. 19; 280(5370:1884-8). For fusion with itstarget cells, HIV-1 uses a trimeric Env complex containing gp120 andgp41 subunits (Burton et al., Nat Immunol. 2004 March; 5(3):233-6). Thefusion potential of the Env complex is triggered by engagement of theCD4 receptor and a coreceptor, usually CCR5 or CXCR4. Neutralizingantibodies seem to work either by binding to the mature trimer on thevirion surface and preventing initial receptor engagement events, or bybinding after virion attachment and inhibiting the fusion process(Parren & Burton, Adv Immunol. 2001; 77:195-262). In the latter case,neutralizing antibodies may bind to epitopes whose exposure is enhancedor triggered by receptor binding. However, given the potential antiviraleffects of neutralizing antibodies, it is not unexpected that HIV-1 hasevolved multiple mechanisms to protect it from antibody binding (Johnson& Desrosiers, Annu Rev Med. 2002; 53:499-518).

Most experimental HIV-1 vaccines tested in human and/or non-humanprimate suggests that a successful vaccine will incorporate immunogensthat elicit broad neutralizing antibodies (bNabs) and robustcell-mediated immunity. HIV-1 envelope glycoprotein (Env) is the mainviral protein involved in the entry of the virus and is also the primarytarget for neutralizing antibodies, but due to immune evasion strategiesand extreme sequence variability of Envs, generation of bNabs has beendaunting task (Phogat S, Wyatt R. Curr Pharm Des. 2007; 13:213-27,Phogat S, et al. J Intern Med. 2007 262:26-43, Karlsson Hedestam G B, etal Nat Rev Microbiol. 2008 6:143-55).

The ability to elicit broad and potent neutralizing antibodies is amajor challenge in the development of an HIV-1 vaccine. Namely, HIV-1has evolved an impressive array of strategies to evade antibody-mediatedneutralization, bNAbs develop over time in a proportion of HIV-1infected individuals, and a handful of broad neutralizing monoclonalantibodies have been isolated from clade B infected donors. Theseantibodies tend to display less breadth and potency against non-clade Bviruses, and they recognize epitopes on the virus that so far havefailed to elicit broad neutralizing responses when incorporated into adiverse range of immunogens.

Recently using a sensitive high-throughput micro-neutralizationscreening of supernatants from approximately 30,000 IgG+ memory B cellsfrom a HIV-1 clade A-infected African donor, two new broadlyneutralizing antibodies PG9 and PG16 that are broad and exceptionallypotent neutralizing antibodies were identified (Walker L, Phogat S, etal. Science. 2009; 326:285-9. Epub 2009 Sep. 3). These antibodiesrecognize a new conserved, yet accessible, vaccine target (consisting ofconserved elements on the variable loops 2 and 3) on the Env and showpreferential binding to HIV Env trimer (model of PG9 and 16 epitopes onHIV-1 trimer.). When tested for binding, these antibodies did not showbinding to many empirically designed soluble (Env gp140) HIV Env trimersthought to be mimics of the native HIV-1 Env spike, suggesting thateither these Env designs are incorrect or they are fixed in a form notrecognized by PG9 and PG16.

Other broadly neutralizing monoclonal antibodies that bind theCD4-binding site have also been identified (Wu et al, Science 329; 856(2010) and Zhou et al., Science. 2010 Aug. 13; 329(5993):811-7. Epub2010 Jul. 8).

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The present invention is based, in part, on novel monoclonal antibodiesidentified from modified HIV-1 envelope (Env) structures, in particular,Env structures with resurfaced stabilized cores (RSC) containingantigenically resurfaced glycoproteins specific for a structurallyconserved site of CD4 receptor binding.

The present invention relates to an isolated or non-naturally occurringhuman monoclonal antibody, wherein the monoclonal antibody mayneutralize a HIV-1 virus in vitro. The monoclonal antibody may beselected from the group consisting of VRC-PG-04 or VRC-PG-05.

The invention also encompasses antibodies that have a heavy chain withthree CDRs which may comprise an amino acid sequence selected from thegroup consisting of the amino acid sequences of VRC-PG-04 or VRC-PG-05of FIG. 8, and a light chain with three CDRs that include an amino acidsequence selected from the group consisting of the amino acid sequencesof VRC-PG-04 or VRC-PG-05 of FIG. 8.

The invention further encompasses compositions that may comprise theisolated or non-naturally occurring anti-HIV antibodies of the presentinvention. The invention also relates to nucleic acid molecules that mayencode the anti-HIV antibodies of the present invention, or a fragmentthereof, vectors that may comprise the nucleic acid molecules that mayencode the anti-HIV antibodies of the present invention, or a fragmentthereof, and cells that may comprise vectors that may comprise thenucleic acid molecules that may encode the anti-HIV antibodies of thepresent invention, or a fragment thereof.

The present invention also relates to methods of immunizing or reducingthe effect of an HIV infection or an HIV-related disease which maycomprise identifying a patient in need of such treatment, andadministering to said patient a therapeutically effective amount of atleast one antibody of the present invention. The method may additionallycomprise the administration of a second therapeutic agent. The secondtherapeutic agent may be an anti-viral agent.

The present invention also relates to methods of immunizing or reducingthe effect of an HIV infection or an HIV-related disease which maycomprise identifying a patient in need of such treatment andadministering to said patient a therapeutically effective amount of: afirst antibody of the present invention, or fragment thereof, specificfor a first epitope which binds to said first antibody and a secondantibody of the present invention, or fragment thereof, specific for asecond epitope which binds to said second antibody.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings, in which:

FIG. 1 depicts protein competition that shows that serum 27-374 hasCD4bs directed neutralizing antibodies.

FIGS. 2A-B depict single cell sort for RSC3 reactive B-cells.

FIG. 3 depicts ELISA Binding of VRC-PG-04 and VRC-PG-05 to severalmutant versions of YU2 gp120 and to the resurfaced stabilized core(RSC3) proteins and its knock out mutant ΔRSC3.

FIG. 4 depicts binding of VRC-PG-04 and VRC-PG-05 to YU2 gp120-basedproteins, HXB2 new core 8b and Du156 gp120 WT by ELISA.

FIGS. 5A-D depict competition ELISA demonstrating that VRC-PG-04 isdirected against CD4bs.

FIGS. 6A-B depict competition ELISA demonstrating that VRC-PG-04, butnot VRC-PG-05, is cross competed by CD4bs antibodies ELISAs done withRSC3 on plate.

FIGS. 7A-B depict competition ELISA using clade B AC10.29 gp1120 onplate and that VRC-PG-05 does not cross-block CD4bs mAbs.

FIGS. 8A-B depicts sequence alignments of VRC-PG antibodies (SEQ ID NOS2-16, respectively, in order of appearance).

FIGS. 9A-B depict antigen binding properties of PGV04. (A) PGV04 bindingto RSC3 (solid lines) and ΔRSC3 (dashed lines), and (B) JRFL gp120 asdetermined by ELISA. Antigens were coated directly on ELISA plates. OD,optical density (absorbance at 405 nm) VRC01, VRC03, b12, and 2G12 wereused as controls for binding to the antigens.

FIGS. 10A-D depict mapping the PGV04 binding epitope. (A) CompetitionELISAs of PGV04 with the CD4bs mAbs b12 and VRC01, CD4-IgG; (B) CD4imAbs 17b and X5; and (C) the glycan binding mAb 2G12 and V3-loop bindingmAb F425. JRFL gp120 was coated on ELISA plates and serial dilutions ofthe mAbs indicated at the top of the graph was added for 30 min at RT.The biotinylated mAbs listed in the legend were then added for 1 hr atRT at EC50 constant concentration. (D) Reverse competition ELISA withserial dilutions of PGV04 binding to JRFL gp120 coated ELISA plates andthen the addition of a constant EC50 concentration of the biotinylatedmAbs listed in the legend. All experiments were performed in duplicate,and data is one representative experiment with SEM plotted.

FIGS. 11A-C depict neutralization activity of PGV04 on a 162- and on a97-virus panel. (A) Potency of neutralization. Boxes are color coded asfollows: orange, median potency between 0.2 and 2.0 μg/ml; and red,median potency <0.2 μg/ml. CRF07_BC, CRF08_BC, and AC viruses were notincluded in the clade analysis, but are counted toward the total numberof neutralized viruses in the 162-virus panel because there was only onevirus tested for each of these clades. Clade D and E viruses were notincluded in the clade analysis, but are counted toward the total numberof neutralized viruses in the 97-virus panel because there was only 2and 3 viruses respectively tested for these clades. (B) Breath ofneutralization. Boxes are colored as follows: orange, 61-90% of virusesneutralized; red, 91-100% of viruses neutralized. As in (A), CRF07_BC,CRF08_BC, AC, D and E viruses were not included in the clade analysis,but are counted toward the total number of neutralized viruses for therespective panel. (C) Dependence of serum NT₅₀ on PGV04. Spearmancorrelation was performed comparing the mAb IC₅₀ and the donor serumNT₅₀ for the 162 viruses tested. The Spearman's rank correlationcoefficient was calculated as −0.71 and the correlation was significantwith a P-value <0.0001. IC₅₀s and NT₅₀s of viruses that did notneutralize were entered at the limit of the assay as 50 μg/ml for themAb or 100 (1/dilution) for the serum.

FIGS. 12A-D depict induction of the co-receptor binding site on gp120and cell surface expressed trimers. Saturating amounts of the mAbslisted in the legend were added to either JRFL or YU2 gp120 coated ELISAplates. After 30 min incubation at RT, a dilution curve of biotinylated(A) 17b or (b) X5 was added for 1 hr at RT. Binding was detected with astreptavidin-AP and absorbance was read at 405 nm. The ability of PGV04to induce the co-receptor binding site on cell surface trimers wasmeasured (C). Saturating amounts of the mAbs listed in the legend wereadded to 293T cells expressing HIV envelope on their surface for 30 min.Either biotinylated 17b or control (D) mAb 2G12 were added to the cells.A streptavidin mAb conjugated to PE was used for detection and bindingwas measured using flow cytometry.

FIGS. 13A-G depict MAb Neutralization and binding to JR-CSF gp120containing single alanine substitutions. (A) PGV04 neutralization ofJR-CSF pseudoviruses containing single alanine substitutions in thegp120 protein. Entry into TZM-bl cells was measured using a luminometerin relative light units (RLU). Neutralization potency relative to WT wascalculated using the following equation: (IC50_WT)/(IC50_mutant)*100.(B) PGV04 binding to JR-CSF gp120 isolated from pseudovirus andcontaining single alanine substitutions. Amino acid numbering is basedon the sequence of HIV-1HXB2. Boxes are color-coded as follows: blue,0-5% neutralization relative wild-type; green, 6-40% neutralizationrelative to wild-type; and yellow, 250-1,000% neutralization relative towild-type.

FIGS. 14A-B depict ability of PGV04 to bind Endo-H and Endo-F-treatedBaL gp120. The mAb binding to (A) mock treated gp120 or (B) Endo-H andEndo-F treated gp120.

FIGS. 15A-I depict (A) Neutralization activity of mAbs against across-clade 162-pseudovirus panel. (B) Neutralization activity of mAbsagainst a cross-clade 97-pseudovirus panel.

FIG. 16 depicts polyreactivity ELISA assay. PGV04 was tested for ELISAreactivity against a panel of antigens. The broad nAbs b12 and 4E10 werealso included for comparison.

FIGS. 17A-D depicts dentification and characterization of mAbs fromHIV-1-infected donors 74 and 0219. (A) RSC3 analysis of serum. Twelvesera from the IAVI Protocol G cohort (donors 17-74) and one serum fromthe CHAVI 001 cohort (donor 0219) were analyzed for RSC3 reduction inserum neutralization on HIV-1 strains JR-FL, PVO.4, YU2 and ZA12.29.Blue bars show the mean serum reduction in neutralization IC50 resultingfrom RSC3 versus ΔRSC3 competition. Sera with greatest reduction werefurther analyzed on HIV-1 strains Q168.a2, RW020.2, Du156.12 andZM109.4. Red bars show the mean reduction on eight viruses. (B) Flowcytometric identification RSC3-reactive IgG+ B cells from donors 74 and0219. Gating and percentage of IgG+ B cells of interest (RSC3+ΔRSC3−)are indicated, with 40 and 26 sorted single B cells from donors 74 and0219 respectively. (C) Protein sequences of heavy and light chainvariable regions of mAbs VRC-PG04 and VRC≈-PG04b, isolated from donor74, and mAbs VRC-CH30-34 isolated from donor 0219. Sequences are alignedto the putative germline ancestral genes and to previously identifiedbroadly neutralizing antibodies VRC01, VRC02 and VRC03. Frameworkregions (FR) and complementarity-determining regions (CDRs) are based onKabat nomenclature (E. A. Kabat, T. T. Wu, K. S. Gottesman, C. Foeller,Sequences of Proteins of Immunological Interest. 5th Edition (1991)).FIG. 17C discloses SEQ ID NOS 17-40, respectively, in order ofappearance. (D) Neutralization dendrograms. VRC-PG04 and VRC-CH31 weretested against genetically diverse Env-pseudoviruses representing themajor HIV-1 clades. Neighbor-joining trees display the protein distanceof gp160 sequences from 178 HIV-1 isolates tested against VRC-PG04 and asubset (80 isolates) tested against VRC-CH31. A scale bar denotes 1%distance in amino acid sequence. Tree branches are colored by theneutralization potencies of VRC-PG04 and VRC-CH31 against eachparticular virus.

FIGS. 18A-C depict a structure of antibodies VRC-PG04 and VRC03 incomplex with HIV-1 gp120. (A) Overall structures. The liganded complexfor the Fab of antibody VRC-PG04 from donor 74 and the HIV-1 gp120envelope glycoprotein from isolate 93TH057 is depicted with polypeptidebackbones in ribbon representation in the left image. The complex of FabVRC03 from donor 45 is depicted in the right image, with surfaces of allvariable domain residues that differ between VRC03 and VRC-PG04 coloredaccording to their chemical characteristics. (B and C) Interactionclose-ups. Critical interactions are shown between the CD4-binding loopof gp120 (purple) and the CDR H2 region of the broadly neutralizingmAbs, VRC03 and VRC-PG04 (reported here) and VRC01 (reported previously(T. Zhou et al., Structural basis for broad and potent neutralization ofHIV-1 by antibody VRC01. Science 329, 811-817 (2010))), with hydrogenbonds depicted as dotted lines. The 1.9 and 2.1 Å resolution structuresof VRC03 and VRC-PG04, respectively, were sufficient to defineinterfacial waters shown in (C), which were unclear in the 2.9 Åstructure of VRC01. The orientation shown in (C) is ˜180° rotated aboutthe vertical axis from the orientation shown in (B).

FIGS. 19A-C depict focused evolution of VRC01-like antibodies. (A)Antibody convergence. The gp120 portions of liganded complexes withVRC01, VRC03 and VRC-PG04 were superimposed to determine the averageantibody per-residue Cα deviation, and the per-residue hydrophobicinteraction (Δ^(i)G) was calculated (E. Krissinel, K. Henrick, Inferenceof macromolecular assemblies from crystalline state. J Mol Biol 372,774-797 (2007)). These two quantities were found to correlate(P-value=0.0427), with antibody residues containing strong hydrophobicinteractions (e.g., at positions 53 and 55 in the heavy chain, and 91and 97 in the light chain, VRC-PG04-relative numbering) displaying highstructural conservation. This correlation is visualized on VRC-PG04 inthe left image, where the ribbon thickness is proportional to thecorresponding per-residue Cα deviation and the paratope surface iscolored according to hydrophobicity, from white (low) to red (high);notably, red surface patches map to thin ribbons. (B) Epitopeconvergence. The HIV-1 gp120 surface involved with CD4 binding containsconformationally invariant regions (e.g. associated with the outerdomain) and conformationally variable regions (e.g. associated with thebridging sheet). Applicants previously hypothesized that theconformationally invariant outer domain-contact for CD4 represents asite of vulnerability (T. Zhou et al., Structural basis for broad andpotent neutralization of HIV-1 by antibody VRC01. Science 329, 811-817(2010)). Applicants analyzed the precision of CD4-binding-site ligandrecognition (vertical axis) versus the IC80 neutralization breadth(horizontal axis) and observed significant correlation (R²=0.6,P-value=0.040). (C) Divergences in sequence and convergences inrecognition. The development of VRC01-like antibodies involves a heavychain derived from the IGHV1-2*02 allele and selected light chain Vκalleles. The far left image depicts ribbon representation model of aputative germline antibody. Somatic hypermutation during the process ofaffinity maturation leads to a divergence in sequence, yet results inthe convergent recognition of similar epitopes. Intersection of theepitope surfaces recognized by VRC01, VRC03 and VRC-PG04 (far rightimage), reveals a remarkable similarity to the site of vulnerability.The primary divergence of this intersection from the hypothesized siteof vulnerability occurs in the region of HIV-1 gp120 recognized by thelight chain of the VRC01-like antibodies. While the separate epitopes ongp120 do show differences in recognition surface, these primarilyinvolve the bridging sheet region, which is likely to adopt a differentconformation in the functional viral spike prior to engagement of CD4.

FIGS. 20A-E depict deep sequencing of expressed heavy and light chainsfrom donors 45 and 74. (A) Heavy and light chain complementation. Theneutralization profiles of VRC01 and VRC03 (donor 45), VRC-PG04 (donor74), and VRCCH31 (donor 0219) and their heavy and light chain chimericswaps are depicted with 20-isolate neutralization dendrograms. Explicitneutralization IC50s are provided in table S13. (B) The repertoire ofheavy chain sequences from donor 45 (2008 sample) and donor 74 (2008sample). Heavy chain sequences are plotted as a function of sequenceidentity to the heavy chain of VRC01 (left), VRC03 (middle) and VRC-PG04(right) and of sequence divergence from putative genomic V_(H)-alleles:upper row plots show sequences of putative IGHV1-2*02 allelic origin;lower row plots show sequences from other allelic origins. Color codingindicates the number of sequences. (C) Repertoire of expressed lightchain sequences from donor 45 (2001 sample). Light chain sequences areplotted as a function of sequence identify to VRC01 (left) and VRC03(right) light chains, and of sequence divergence from putative genomicV-gene alleles. Sequences with 2-residue deletions in the CDR L1 region(which is observed in VRC01 and VRC03) are shown as black dots. Twolight chain sequences, with 92.0% identify to VRC01 (sequence ID 181371)and with 90.3% identify to VRC03 (sequence ID 223454) are highlightedwith red triangles. (D) Functional assessment of light chain sequencesidentified by deep sequencing. The neutralization profiles of sequence181371 reconstituted with the VRC01 heavy chain (named gVRC-L1_(d45))and of sequence 223454 reconstituted with the VRC03 heavy chain (namedgVRC-L2_(d45)) are depicted with 20-isolate neutralization dendrograms;explicit neutralization IC₅₀s are shown provided in table S22. (E)Functional assessment of heavy chain sequences identified by deepsequencing. Heavy chain sequences from donors 45 and 74 were synthesizedand expressed with either the light chain of VRC01 or VRC03 (for donor45) or the light chain of VRC-PG04 (for donor 74) and evaluated forneutralization. Neutralizing sequences are shown as red stars and arelabeled. gVRC-H(n)_(d74) refers to the heavy chains with confirmedneutralization when reconstituted with the light chain of VRC-PG04, withcontrols. Applicants also assessed 454-derived sequences for structuralcompatibility with the VRC01, VRC03, and VRC-PG04 gp120-complex crystalstructures using a threading algorithm which assessed structuralcompatibility using the DFIRE statistical potential (H. Zhou, Y. Zhou,Distance-scaled, finite ideal-gas reference state improvesstructure-derived potentials of mean force for structure selection andstability prediction. Protein Sci 11, 2714-2726 (2002)). None of the tensequences with optimal DFIRE scores, nor those with high germlinedivergence of non-IGHV1-2*02 genomic origin displayed neutralizationwhen reconstituted with the VRC01 light chain (FIG. 4E). Thus, sequencesimilarity, IGHV1-2*02 origin, and divergence all correlate withneutralization potential, but other factors such as predicted structuralcompatibility failed to identify VRC01-like antibodies

FIGS. 21A-C depict maturational similarities of VRC01-like antibodies indifferent donors revealed by cross-donor phylogenetic analysis. (A)Maximum-likelihood trees of heavy chain sequences of the IGHV1-2*02origin from donor 45 (left) and donor 74 (right). The subset ofsequences shown was selected based on the germline divergence asdescribed in SOM. The donor 45 tree is rooted by the putative revertedunmutated ancestor of the heavy chain of VRC01, and also includesspecific neutralizing sequences from donor 74 and 0219 (shown in red).Similarly the donor 74 tree is rooted in the putative reverted unmutatedancestor of the heavy chain of VRC-PG04, and sequences donor 45 and 0219are included in the cross-donor phylogenetic analysis. Bars representing0.1 changes per nucleotide site are shown. Insets show J chainassignments for all sequences within the neutralizing subtree identifiedby an iterative neighbor-joining tree analysis as described in SOM. (B)Phylogenetically inferred maturation intermediates. Backbone ribbonrepresentations are shown for HIV-1 gp120 (red) and the heavy chainvariable domains (green). Critical intermediates inferred from thephylogenetic tree in (A) are labeled I_(d45), II_(d45), III_(d45),I_(d74) and II_(d74). The number of V_(H)-gene mutations is provided(e.g. for the 23 mutations associated with the first intermediation ofdonor 45, “I_(d45): 23”), and the location of these is highlighted inthe surface representation and colored according to their chemistry.

FIGS. 22A-E depict an analysis of the heavy chain antibodyome of donor74 and identification of heavy chains with HIV-1 neutralizing activity.Identity/divergence-grid analysis, cross-donor phylogenetic analysis,and CDR H3 analysis were coupled to functional characterization ofselected heavy chain sequences. This provides a means for identificationof novel heavy chains with HIV-1 neutralizing activity. (A)Identity/divergence-grid analysis. The location of the 63 synthesizedIGHV1-2*02 heavy chains from donor 74 is shown, including neutralizing(red stars) and non-neutralizing (black stars) sequences. (B)Cross-donor phylogenetic analysis and CDR H3 lineage analysis. Amaximum-likelihood tree of the 70 synthesized heavy chain sequences(including 7 non-IGHV1-2*02 sequences) is rooted at the putativereverted unmutated ancestor of VRC-PG04. The probe-identified VRC-PG andVRC-CH antibodies are shown in red text along with the 24 genomicallyidentified heavy chain sequences, gVRC-H(1-24)_(d74), which were foundto neutralize HIV-1 when reconstituted with the light chain of VRC-PG04.Grid locations and CDR H3 classes are specified for neutralizing andnon-neutralizing sequences. Within each CDR H3 class, all sequences withidentical CDR H3s are highlighted in orange in the far right grids (withthe number of total sequences corresponding to each CDR H3 class shown).(C) Expression levels of selected heavy chains reconstituted with thelight chain of VRC-PG04 versus breadth of neutralization. (D)Neutralization potency of reconstituted cross-donor phylogeny-predictedantibodies on seven HIV-1 isolates. (E) CDR H3 analysis of donor 74heavy chain sequences. For each of the 110,386 sequences derived fromthe IGHV1-2*02 allele, the CDR H3 was determined and its percentidentity to that of the VRC-PG04 heavy chain was color coded as shown,and graphed. The sequences with high CDR H3 identity to VRC-PG04 residein regions of high overall heavy chain sequence identity, even forsequences with a low divergence from IGHV1-2*02.

DETAILED DESCRIPTION

The present invention provides a novel method for isolating novel broadand potent neutralizing monoclonal antibodies against HIV. Inparticular, the search began with an IAVI-sponsored clinical studycalled Protocol G, a global hunt for new broadly neutralizing antibodiesagainst HIV. Blood samples were collected from more than 1,800HIV-positive people across the world. The method involves selection of aPBMC donor with high neutralization titer of antibodies in the plasma. Bcells are screened for neutralization activity prior to rescue ofantibodies. A process that more accurately predicted whether a givensample contained broadly neutralizing antibodies was developed and thesamples were scored in terms of how many types of HIV they neutralized,and the top 10% were separated for further study. Novel neutralizingantibodies are obtained by emphasizing neutralization as the initialscreen (see, e.g., Walker L, Phogat S, et al. Science. 2009; 326:285-9.Epub 2009 Sep. 3).

In an advantageous embodiment, the recombinant rescue of the monoclonalantibodies involves use of antigen-specific B-cell sorting as describedby Wu et al (Science 329; 856 (2010)). To isolate CD4bs-directed mAbs,Applicants used a recently described method of antigen-specific memoryB-cell sorting (Scheid et al., Nature 458, 636 (2009)), together withsingle cell PCR, to amplify IgG heavy and light chain genes from thecDNA of individual B cells (Scheid et al., Nature 458, 636 (2009),Wrammert et al., Nature 453, 667 (2008)). RSC3 and ΔRSC3 were expressedwith a tagged amino acid sequence that allows biotin labeling (Wu etal., Science 329; 856 (2010)). The two proteins could thus bedistinguished by FACS analysis after labeling with streptavidin (SA)conjugated to the fluorochromes allophycocyanin (SA-APC) orphycoerythrin (SA-PE), respectively. Peripheral blood mononuclear cells(PBMC) were incubated with RSC3 SA-APC and ΔRSC3 SA-PE, and singleantigen-specific memory B cells were sorted into wells of a microtiterplate after selecting for memory B cells (CD19+, CD20+, IgG+) that boundto the RSC3 but not ΔRSC3 probe. RSC3-specific memory B cells weresorted and the matching heavy and light chain genes were successfullyamplified from 12 cells. After cloning into IgG1 expression vectors thatreconstituted the heavy and light chain constant regions, the full IgGmAbs were expressed.

In another embodiment, the recombinant rescue of the monoclonalantibodies involves use of a B cell culture system as described inWeitcamp J-H et al., J. Immunol. 171:4680-4688 (2003). Any other methodfor rescue of single B cells clones known in the art also may beemployed such as EBV immortalization of B cells (Traggiai E., et al.,Nat. Med. 10(8):871-875 (2004)), electrofusion (Buchacher, A., et al.,1994. AIDS Res. Hum. Retroviruses 10:359-369), and B cell hybridoma(Karpas A. et al., Proc. Natl. Acad. Sci. USA 98:1799-1804 (2001).

In some embodiments, monoclonal antibodies were rescued from the B cellcultures using variable chain gene-specific RT-PCR, and transfectantwith combinations of H and L chain clones were screened again forneutralization and HIV antigen binding activities. mAbs withneutralization properties were selected for further characterization.

The antibodies of the present invention were identified according tothese methods are disclosed by Wu et al, Science 329; 856 (2010). Theseantibodies were isolated from a human sample obtained through IAVI'sProtocol G, and are referred to as VRC-PG-04 or VRC-PG-05—theseantibodies neutralize HIV in vitro.

The invention is based on novel monoclonal antibodies and antibodyfragments that neutralize HIV infection. In some embodiments, thesemonoclonal antibodies and antibody fragments have a particularly highpotency in neutralizing HIV infection in vitro across multiple clades.Such antibodies are desirable, as only low concentrations are requiredin order to neutralize a given amount of virus. This facilitates higherlevels of protection while administering lower amounts of antibody.Human monoclonal antibodies that secrete such antibodies are alsoincluded within the scope of the invention.

The invention also relates to various methods and uses involving theantibodies of the invention and the epitopes to which they bind.

The invention provides novel monoclonal or recombinant antibodies havingparticularly high potency in neutralizing HIV. The invention alsoprovides fragments of these recombinant or monoclonal antibodies,particularly fragments that retain the antigen-binding activity of theantibodies, for example which retain at least one complementaritydetermining region (CDR) specific for HIV proteins. In thisspecification, by “high potency in neutralizing HIV” is meant that anantibody molecule of the invention neutralizes HIV in a standard assayat a concentration lower than antibodies known in the art.

The antibody molecule of the present invention may have concentrationsof less than about 1 μg/ml, between about 1-10 μg/ml or greater thanabout 10 μg/ml to achieve 50% or 80% neutralization. In an exemplaryembodiment, the antibody molecule of the present invention may have theconcentrations of Table 4 which represent the monoclonal antibodyconcentration required to achieve 50% (Table 4A) or 80% (Table 4B)neutralization.

In another embodiment, the antibody molecule of the present inventionmay neutralize at a concentration of 0.16 μg/ml or lower (i.e. 0.15,0.125, 0.1, 0.075, 0.05, 0.025, 0.02, 0.016, 0.015, 0.0125, 0.01,0.0075, 0.005, 0.004 or lower), preferably 0.016 μg/ml or lower (anantibody concentration of 10-8 or lower, preferably 10-9 M or lower,preferably 10-10 M or lower, i.e. 10-11 M, 10-12 M, 10-13 M or lower).This means that only very low concentrations of antibody are requiredfor 50% neutralization of a clinical isolate of HIV in vitro. Potencycan be measured using a standard neutralization assay as described inthe art.

The antibodies of the invention are able to neutralize HIV. Monoclonalantibodies can be produced by known procedures, e.g., as described by R.Kennet et al. in “Monoclonal Antibodies and Functional Cell Lines;Progress and Applications”. Plenum Press (New York), 1984. Furthermaterials and methods applied are based on known procedures, e.g., suchas described in J. Virol. 67:6642-6647, 1993.

These antibodies can be used as prophylactic or therapeutic agents uponappropriate formulation, or as a diagnostic tool.

A “neutralizing antibody” is one that can neutralize the ability of thatpathogen to initiate and/or perpetuate an infection in a host and/or intarget cells in vitro. The invention provides a neutralizing monoclonalhuman antibody, wherein the antibody recognizes an antigen from HIV.

Preferably an antibody according to the invention is a novel monoclonalantibody referred to herein as VRC-PG-04 or VRC-PG-05. These antibodieswere initially isolated from human samples obtained from IAVI's ProtocolG. These antibodies have been shown to neutralize HIV in vitro.

The CDRs of the antibody heavy chains are referred to as CDRH1, CDRH2and CDRH3, respectively. Similarly, the CDRs of the antibody lightchains are referred to as CDRL1, CDRL2 and CDRL3, respectively. Theposition of the CDR amino acids are defined according to the IMGTnumbering system as: CDR1—IMGT positions 27 to 38, CDR2—IMGT positions56 to 65 and CDR3—IMGT positions 105 to 117. (Lefranc, M P. et al. 2003IMGT unique numbering for immunoglobulin and T cell receptor variabledomains and Ig superfamily V-like domains. Dev Comp Immunol.27(1):55-77; Lefranc, M P. 1997. Unique database numbering system forimmunogenetic analysis. Immunology Today, 18:509; Lefranc, M P. 1999.The IMGT unique numbering for Immunoglobulins, T cell receptors andIg-like domains. The Immunologist, 7:132-136.)

The amino acid sequences of the CDR3 regions of the light and heavychains of the antibodies are shown in FIG. 8.

As used herein, a neutralizing antibody may inhibit the entry of HIV-1virus with a neutralization index >1.5 or >2.0. Broad and potentneutralizing antibodies may neutralize greater than about 50% of HIV-1viruses (from diverse clades and different strains within a clade) in aneutralization assay.

Assays for screening for neutralizing antibodies are known in the art. Aneutralization assay approach has been described previously (Binley J M,et al., (2004). Comprehensive Cross-Clade Neutralization Analysis of aPanel of Anti-Human Immunodeficiency Virus Type 1 Monoclonal Antibodies.J. Virol. 78: 13232-13252). Pseudotyped viruses may be generated byco-transfecting cells with at least two plasmids encoding the solubleEnv cDNA of the present invention and the rest of the HIV genomeseparately. In the HIV genome encoding vector, the Env gene may bereplaced by the firefly luciferase gene. Transfectant supernatantscontaining pseudotyped virus may be co-incubated overnight with B cellsupernatants derived from activation of an infected donor's primaryperipheral blood mononuclear cells (PBMCs). Cells stably transfectedwith and expressing CD4 plus the CCR5 and CXCR4 coreceptors may be addedto the mixture and incubated for 3 days at 37° C. Infected cells may bequantified by luminometry.

The neutralization index may be expressed as the ratio of normalizedrelative luminescence units (RLU) of the test viral strain to that of acontrol virus derived from the same test B cell culture supernatant. Thecut-off values used to distinguish neutralizing hits may be determinedby the neutralization index of a large number of “negative controlwells” containing B cell culture supernatants derived from healthydonors. Such a method was successful for the isolation andcharacterization of PG9 and PG16.

The method of U.S. Pat. No. 7,386,232 may also be utilized for thescreening of broad neutralizing antibodies. An envelope-enzyme fusionprotein may be constructed by attaching an enzyme to the C-terminal endof an envelope protein. Virus particles which may comprise the fusionprotein and wild type and/or soluble envelope glycoprotein may begenerated and used to infect target cells in the presence of a patients'sera. Activities of enzyme measured in such infected cells are measuresof virus binding and entry to the target cells that are mediated by thewild type viral envelope protein. Examples of enzymes that can be usedto generate the fusion protein include, but are not limited to,luciferase, bacterial or placental alkaline phosphatase,β-galactosidase, and fluorescent proteins such as Green fluorescentprotein or toxins. The assay, in general, can also be carried out in96-well plate. Decreased enzyme activities in the presence of the seraindicate that there are neutralizing antibodies in the sera.

In an advantageous embodiment, VRC-PG-04 and VRC-PG-05 were isolatedusing antigen specific B-cell sorting and PCR amplification of heavy andlight chain genes as described by Wu et al (Science 329; 856 (2010)).Epitope specific protein probes (RSC3) and knock out mutant (delta RSC3)were utilized as described by Wu et al (Science 329; 856 (2010)).

In another embodiment to isolate CD4bs-directed mAbs, a method ofantigen-specific memory B-cell sorting (Wu et al (Science 329; 856(2010))), together with single cell PCR, to amplify IgG heavy and lightchain genes from the cDNA of individual B cells (J. F. Scheid et al.,Broad diversity of neutralizing antibodies isolated from memory B cellsin HIV-infected individuals. Nature 458, 636 (2009) and J. Wrammert etal., Nature 453, 667 (2008)) is preferred. Mutant Env probes areexpressed with a tagged amino acid sequence that allows biotin labelingto distinguish them by FACS analysis after labeling with streptavidin(SA) conjugated to the fluorochromes allophycocyanin (SA-APC) orphycoerythrin (SA-PE), respectively. Peripheral blood mononuclear cells(PBMC) from a donor are incubated with the labeled mutant Env probes,and single antigen-specific memory B cells were sorted into wells of amicrotiter plate after selecting for memory B cells (CD19+, CD20+, IgG+)that bind to the reference probe. The reference-probe-specific memory Bcells are sorted and the matching heavy and light chain genes areamplified. After cloning into IgG1 expression vectors that reconstitutethe heavy and light chain constant regions, the full IgG mAbs areexpressed.

The cloning step for separating individual clones from the mixture ofpositive cells may be carried out using limiting dilution,micromanipulation, single cell deposition by cell sorting or anothermethod known in the art. Preferably the cloning is carried out usinglimiting dilution.

The immortalized B cell clones of the invention can be used in variousways e.g. as a source of monoclonal antibodies, as a source of nucleicacid (DNA or mRNA) encoding a monoclonal antibody of interest, forresearch, etc.

The epitopes recognized by these antibodies may have a number of uses.The epitopes and mimotopes in purified or synthetic form can be used toraise immune responses (i.e. as a vaccine, or for the production ofantibodies for other uses) or for screening patient serum for antibodiesthat immunoreact with the epitopes or mimotopes. Preferably, such anepitope or mimotope, or antigen which may comprise such an epitope ormimotope is used as a vaccine for raising an immune response. Theantibodies of the invention can also be used in a method to monitor thequality of vaccines in particular to check that the antigen in a vaccinecontains the correct immunogenic epitope in the correct conformation.

The epitopes may also be useful in screening for ligands that bind tosaid epitopes. Such ligands preferably block the epitopes and thusprevent infection.

Compounds which have a chemical structure selected using the invention,wherein said compounds are neutralizing antibody binders, form a furtheraspect of the invention; and, such compounds may be used in methods ofmedical treatments, such as for diagnosis, preventing or treating HIV orfor eliciting antibodies for diagnosis of HIV, including use invaccines. Further, such compounds may be used in the preparation ofmedicaments for such treatments or prevention, or compositions fordiagnostic purposes. The compounds may be employed alone or incombination with other treatments, vaccines or preventatives; and, thecompounds may be used in the preparation of combination medicaments forsuch treatments or prevention, or in kits containing the compound andthe other treatment or preventative.

The terms “protein”, “peptide”, “polypeptide”, and “amino acid sequence”are used interchangeably herein to refer to polymers of amino acidresidues of any length. The polymer may be linear or branched, it maycomprise modified amino acids or amino acid analogs, and it may beinterrupted by chemical moieties other than amino acids. The terms alsoencompass an amino acid polymer that has been modified naturally or byintervention; for example disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling or bioactivecomponent.

As used herein, the terms “antigen” or “immunogen” are usedinterchangeably to refer to a substance, typically a protein, which iscapable of inducing an immune response in a subject. The term alsorefers to proteins that are immunologically active in the sense thatonce administered to a subject (either directly or by administering tothe subject a nucleotide sequence or vector that encodes the protein) isable to evoke an immune response of the humoral and/or cellular typedirected against that protein.

The term “antibody” includes intact molecules as well as fragmentsthereof, such as Fab, F(ab′)₂, Fv and scFv which are capable of bindingthe epitope determinant. These antibody fragments retain some ability toselectively bind with its antigen or receptor and include, for example:

-   -   (i) Fab, the fragment which contains a monovalent        antigen-binding fragment of an antibody molecule can be produced        by digestion of whole antibody with the enzyme papain to yield        an intact light chain and a portion of one heavy chain;    -   (ii) Fab′, the fragment of an antibody molecule can be obtained        by treating whole antibody with pepsin, followed by reduction,        to yield an intact light chain and a portion of the heavy chain;        two Fab′ fragments are obtained per antibody molecule;    -   (iii) F(ab′)2, the fragment of the antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragments        held together by two disulfide bonds;    -   (iv) scFv, including a genetically engineered fragment        containing the variable region of a heavy and a light chain as a        fused single chain molecule.

General methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), which is incorporated herein byreference).

A “neutralizing antibody” may inhibit the entry of HIV-1 virus forexample SF162 and/or JRCSF with a neutralization index >1.5 or >2.0.Broad and potent neutralizing antibodies may neutralize greater thanabout 50% of HIV-1 viruses (from diverse clades and different strainswithin a clade) in a neutralization assay. The inhibitory concentrationof the monoclonal antibody may be less than about 25 mg/ml to neutralizeabout 50% of the input virus in the neutralization assay.

An “isolated antibody” or “non-naturally occurring antibody” is one thathas been separated and/or recovered from a component of its naturalenvironment. Contaminant components of its natural environment arematerials that would interfere with diagnostic or therapeutic uses forthe antibody, and may include enzymes, hormones, and other proteinaceousor nonproteinaceous solutes. In preferred embodiments, the antibody ispurified: (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight; (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator; or (3)to homogeneity by SDS-PAGE under reducing or non-reducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies which may comprise the population areidentical except for possible naturally occurring mutations that may bepresent in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto polyclonal antibody preparations that include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Inaddition to their specificity, the monoclonal antibodies areadvantageous in that they may be synthesized uncontaminated by otherantibodies. The modifier “monoclonal” is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies useful in the present invention maybe prepared by the hybridoma methodology first described by Kohler etal., Nature, 256:495 (1975), or may be made using recombinant DNAmethods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S.Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolatedfrom phage antibody libraries using the techniques described in Clacksonet al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991), for example.

An “antibody fragment” may comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870;Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments.

It should be understood that the proteins, including the antibodies ofthe invention may differ from the exact sequences illustrated anddescribed herein. Thus, the invention contemplates deletions, additionsand substitutions to the sequences shown, so long as the sequencesfunction in accordance with the methods of the invention. In thisregard, particularly preferred substitutions will generally beconservative in nature, i.e., those substitutions that take place withina family of amino acids. For example, amino acids are generally dividedinto four families: (1) acidic—aspartate and glutamate; (2)basic—lysine, arginine, histidine; (3) non-polar—alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar—glycine, asparagine, glutamine, cysteine, serinethreonine, tyrosine. Phenylalanine, tryptophan, and tyrosine aresometimes classified as aromatic amino acids. It is reasonablypredictable that an isolated or non-naturally occurring replacement ofleucine with isoleucine or valine, or vice versa; an aspartate with aglutamate or vice versa; a threonine with a serine or vice versa; or asimilar conservative replacement of an amino acid with a structurallyrelated amino acid, will not have a major effect on the biologicalactivity. Proteins having substantially the same amino acid sequence asthe sequences illustrated and described but possessing minor amino acidsubstitutions that do not substantially affect the immunogenicity of theprotein are, therefore, within the scope of the invention.

As used herein the terms “nucleotide sequences” and “nucleic acidsequences” refer to deoxyribonucleic acid (DNA) or ribonucleic acid(RNA) sequences, including, without limitation, messenger RNA (mRNA),DNA/RNA hybrids, or synthetic nucleic acids. The nucleic acid can besingle-stranded, or partially or completely double-stranded (duplex).Duplex nucleic acids can be homoduplex or heteroduplex.

As used herein the term “transgene” may used to refer to “recombinant”nucleotide sequences that may be derived from any of the nucleotidesequences encoding the proteins of the present invention. The term“recombinant” means a nucleotide sequence that has been manipulated “byman” and which does not occur in nature, or is linked to anothernucleotide sequence or found in a different arrangement in nature. It isunderstood that manipulated “by man” means manipulated by someartificial means, including by use of machines, codon optimization,restriction enzymes, etc.

For example, in one embodiment the nucleotide sequences may be mutatedsuch that the activity of the encoded proteins in vivo is abrogated. Inanother embodiment the nucleotide sequences may be codon optimized, forexample the codons may be optimized for human use. In preferredembodiments the nucleotide sequences of the invention are both mutatedto abrogate the normal in vivo function of the encoded proteins, andcodon optimized for human use. For example, each of the Gag, Pol, Env,Nef, RT, and Int sequences of the invention may be altered in theseways.

As regards codon optimization, the nucleic acid molecules of theinvention have a nucleotide sequence that encodes the antigens of theinvention and can be designed to employ codons that are used in thegenes of the subject in which the antigen is to be produced. Manyviruses, including HIV and other lentiviruses, use a large number ofrare codons and, by altering these codons to correspond to codonscommonly used in the desired subject, enhanced expression of theantigens can be achieved. In a preferred embodiment, the codons used are“humanized” codons, i.e., the codons are those that appear frequently inhighly expressed human genes (Andre et al., J. Virol. 72:1497-1503,1998) instead of those codons that are frequently used by HIV. Suchcodon usage provides for efficient expression of the transgenic HIVproteins in human cells. Any suitable method of codon optimization maybe used. Such methods, and the selection of such methods, are well knownto those of skill in the art. In addition, there are several companiesthat will optimize codons of sequences, such as Geneart (geneart.com).Thus, the nucleotide sequences of the invention can readily be codonoptimized.

The invention further encompasses nucleotide sequences encodingfunctionally and/or antigenically equivalent variants and derivatives ofthe antigens of the invention and functionally equivalent fragmentsthereof. These functionally equivalent variants, derivatives, andfragments display the ability to retain antigenic activity. Forinstance, changes in a DNA sequence that do not change the encoded aminoacid sequence, as well as those that result in conservativesubstitutions of amino acid residues, one or a few amino acid deletionsor additions, and substitution of amino acid residues by amino acidanalogs are those which will not significantly affect properties of theencoded polypeptide. Conservative amino acid substitutions areglycine/alanine; valine/isoleucine/leucine; asparagine/glutamine;aspartic acid/glutamic acid; serine/threonine/methionine;lysine/arginine; and phenylalanine/tyrosine/tryptophan. In oneembodiment, the variants have at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% homology oridentity to the antigen, epitope, immunogen, peptide or polypeptide ofinterest.

For the purposes of the present invention, sequence identity or homologyis determined by comparing the sequences when aligned so as to maximizeoverlap and identity while minimizing sequence gaps. In particular,sequence identity may be determined using any of a number ofmathematical algorithms. A nonlimiting example of a mathematicalalgorithm used for comparison of two sequences is the algorithm ofKarlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268,modified as in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993; 90:5873-5877.

Another example of a mathematical algorithm used for comparison ofsequences is the algorithm of Myers & Miller, CABIOS 1988; 4: 11-17.Such an algorithm is incorporated into the ALIGN program (version 2.0)which is part of the GCG sequence alignment software package. Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used. Yet another useful algorithm for identifying regions oflocal sequence similarity and alignment is the FASTA algorithm asdescribed in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85:2444-2448.

Advantageous for use according to the present invention is the WU-BLAST(Washington University BLAST) version 2.0 software. WU-BLAST version 2.0executable programs for several UNIX platforms can be downloaded fromftp://blast.wustl.edu/blast/executables. This program is based onWU-BLAST version 1.4, which in turn is based on the public domainNCBI-BLAST version 1.4 (Altschul & Gish, 1996, Local alignmentstatistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschulet al., Journal of Molecular Biology 1990; 215: 403-410; Gish & States,1993; Nature Genetics 3: 266-272; Karlin & Altschul, 1993; Proc. Natl.Acad. Sci. USA 90: 5873-5877; all of which are incorporated by referenceherein).

The various recombinant nucleotide sequences and antibodies of theinvention are made using standard recombinant DNA and cloningtechniques. Such techniques are well known to those of skill in the art.See for example, “Molecular Cloning: A Laboratory Manual”, secondedition (Sambrook et al. 1989).

The nucleotide sequences of the present invention may be inserted into“vectors.” The term “vector” is widely used and understood by those ofskill in the art, and as used herein the term “vector” is usedconsistent with its meaning to those of skill in the art. For example,the term “vector” is commonly used by those skilled in the art to referto a vehicle that allows or facilitates the transfer of nucleic acidmolecules from one environment to another or that allows or facilitatesthe manipulation of a nucleic acid molecule.

Any vector that allows expression of the antibodies of the presentinvention may be used in accordance with the present invention. Incertain embodiments, the antibodies of the present invention may be usedin vitro (such as using cell-free expression systems) and/or in culturedcells grown in vitro in order to produce the encoded HIV-antibodieswhich may then be used for various applications such as in theproduction of proteinaceous vaccines. For such applications, any vectorthat allows expression of the antibodies in vitro and/or in culturedcells may be used.

For applications where it is desired that the antibodies be expressed invivo, for example when the transgenes of the invention are used in DNAor DNA-containing vaccines, any vector that allows for the expression ofthe antibodies of the present invention and is safe for use in vivo maybe used. In preferred embodiments the vectors used are safe for use inhumans, mammals and/or laboratory animals.

For the antibodies of the present invention to be expressed, the proteincoding sequence should be “operably linked” to regulatory or nucleicacid control sequences that direct transcription and translation of theprotein. As used herein, a coding sequence and a nucleic acid controlsequence or promoter are said to be “operably linked” when they arecovalently linked in such a way as to place the expression ortranscription and/or translation of the coding sequence under theinfluence or control of the nucleic acid control sequence. The “nucleicacid control sequence” can be any nucleic acid element, such as, but notlimited to promoters, enhancers, IRES, introns, and other elementsdescribed herein that direct the expression of a nucleic acid sequenceor coding sequence that is operably linked thereto. The term “promoter”will be used herein to refer to a group of transcriptional controlmodules that are clustered around the initiation site for RNA polymeraseII and that when operationally linked to the protein coding sequences ofthe invention lead to the expression of the encoded protein. Theexpression of the transgenes of the present invention can be under thecontrol of a constitutive promoter or of an inducible promoter, whichinitiates transcription only when exposed to some particular externalstimulus, such as, without limitation, antibiotics such as tetracycline,hormones such as ecdysone, or heavy metals. The promoter can also bespecific to a particular cell-type, tissue or organ. Many suitablepromoters and enhancers are known in the art, and any such suitablepromoter or enhancer may be used for expression of the transgenes of theinvention. For example, suitable promoters and/or enhancers can beselected from the Eukaryotic Promoter Database (EPDB).

The vectors used in accordance with the present invention shouldtypically be chosen such that they contain a suitable gene regulatoryregion, such as a promoter or enhancer, such that the antibodies of theinvention can be expressed.

For example, when the aim is to express the antibodies of the inventionin vitro, or in cultured cells, or in any prokaryotic or eukaryoticsystem for the purpose of producing the protein(s) encoded by thatantibody, then any suitable vector can be used depending on theapplication. For example, plasmids, viral vectors, bacterial vectors,protozoal vectors, insect vectors, baculovirus expression vectors, yeastvectors, mammalian cell vectors, and the like, can be used. Suitablevectors can be selected by the skilled artisan taking into considerationthe characteristics of the vector and the requirements for expressingthe antibodies under the identified circumstances.

In an advantageous embodiment, IgG1 expression vectors may be utilizedto reconstitute heavy and light chain constant regions if heavy andlight chain genes of the antibodies of the present invention are cloned.

When the aim is to express the antibodies of the invention in vivo in asubject, for example in order to generate an immune response against anHIV-1 antigen and/or protective immunity against HIV-1, expressionvectors that are suitable for expression on that subject, and that aresafe for use in vivo, should be chosen. For example, in some embodimentsit may be desired to express the antibodies of the invention in alaboratory animal, such as for pre-clinical testing of the HIV-1immunogenic compositions and vaccines of the invention. In otherembodiments, it will be desirable to express the antibodies of theinvention in human subjects, such as in clinical trials and for actualclinical use of the immunogenic compositions and vaccine of theinvention. Any vectors that are suitable for such uses can be employed,and it is well within the capabilities of the skilled artisan to selecta suitable vector. In some embodiments it may be preferred that thevectors used for these in vivo applications are attenuated to vectorfrom amplifying in the subject. For example, if plasmid vectors areused, preferably they will lack an origin of replication that functionsin the subject so as to enhance safety for in vivo use in the subject.If viral vectors are used, preferably they are attenuated orreplication-defective in the subject, again, so as to enhance safety forin vivo use in the subject.

In preferred embodiments of the present invention viral vectors areused. Viral expression vectors are well known to those skilled in theart and include, for example, viruses such as adenoviruses,adeno-associated viruses (AAV), alphaviruses, herpesviruses,retroviruses and poxviruses, including avipox viruses, attenuatedpoxviruses, vaccinia viruses, and particularly, the modified vacciniaAnkara virus (MVA; ATCC Accession No. VR-1566). Such viruses, when usedas expression vectors are innately non-pathogenic in the selectedsubjects such as humans or have been modified to render themnon-pathogenic in the selected subjects. For example,replication-defective adenoviruses and alphaviruses are well known andcan be used as gene delivery vectors.

The nucleotide sequences and vectors of the invention can be deliveredto cells, for example if the aim is to express the HIV-1 antigens incells in order to produce and isolate the expressed proteins, such asfrom cells grown in culture. For expressing the antibodies in cells anysuitable transfection, transformation, or gene delivery methods can beused. Such methods are well known by those skilled in the art, and oneof skill in the art would readily be able to select a suitable methoddepending on the nature of the nucleotide sequences, vectors, and celltypes used. For example, transfection, transformation, microinjection,infection, electroporation, lipofection, or liposome-mediated deliverycould be used. Expression of the antibodies can be carried out in anysuitable type of host cells, such as bacterial cells, yeast, insectcells, and mammalian cells. The antibodies of the invention can also beexpressed using including in vitro transcription/translation systems.All of such methods are well known by those skilled in the art, and oneof skill in the art would readily be able to select a suitable methoddepending on the nature of the nucleotide sequences, vectors, and celltypes used.

In preferred embodiments, the nucleotide sequences, antibodies of theinvention are administered in vivo, for example where the aim is toproduce an immunogenic response in a subject. A “subject” in the contextof the present invention may be any animal. For example, in someembodiments it may be desired to express the transgenes of the inventionin a laboratory animal, such as for pre-clinical testing of the HIV-1immunogenic compositions and vaccines of the invention. In otherembodiments, it will be desirable to express the antibodies of theinvention in human subjects, such as in clinical trials and for actualclinical use of the immunogenic compositions and vaccine of theinvention. In preferred embodiments the subject is a human, for examplea human that is infected with, or is at risk of infection with, HIV-1.

The term “pharmaceutical composition” is used herein to define a solidor liquid composition in a form, concentration and level of puritysuitable for administration to a patient (e.g. a human patient) uponwhich administration it can elicit the desired physiological changes.The terms “immunogenic composition” and “immunological composition” and“immunogenic or immunological composition” cover any composition thatelicits an immune response against the targeted pathogen, HIV. Termssuch as “vaccinal composition” and “vaccine” and “vaccine composition”cover any composition that induces a protective immune response againstthe targeted pathogen or which efficaciously protects against thepathogen; for instance, after administration or injection, elicits aprotective immune response against the targeted pathogen or providesefficacious protection against the pathogen. Accordingly, an immunogenicor immunological composition induces an immune response which can, butneed not, be a protective immune response. An immunogenic orimmunological composition can be used in the treatment of individualsinfected with the pathogen, e.g., to stimulate an immune responseagainst the pathogen, such as by stimulating antibodies against thepathogen. Thus, an immunogenic or immunological composition can be apharmaceutical composition. Furthermore, when the text speaks of“immunogen, antigen or epitope”, an immunogen can be an antigen or anepitope of an antigen. A diagnostic composition is a compositioncontaining a compound or antibody, e.g., a labeled compound or antibody,that is used for detecting the presence in a sample, such as abiological sample, e.g., blood, semen, vaginal fluid, etc, of anantibody that binds to the compound or an immunogen, antigen or epitopethat binds to the antibody; for instance, an anti-HIV antibody or an HIVimmunogen, antigen or epitope.

For such in vivo applications the nucleotide sequences, antibodies ofthe invention are preferably administered as a component of animmunogenic composition which may comprise the nucleotide sequencesand/or antigens of the invention in admixture with a pharmaceuticallyacceptable carrier. The immunogenic compositions of the invention areuseful to stimulate an immune response against HIV-1 and may be used asone or more components of a prophylactic or therapeutic vaccine againstHIV-1 for the prevention, amelioration or treatment of AIDS. The nucleicacids and vectors of the invention are particularly useful for providinggenetic vaccines, i.e. vaccines for delivering the nucleic acidsencoding the antibodies of the invention to a subject, such as a human,such that the antibodies are then expressed in the subject to elicit animmune response.

The present invention also relates to methods of immunizing or reducingthe effect of an HIV infection or an HIV related disease which maycomprise identifying a patient in need of such treatment, andadministering to said patient a therapeutically effective amount of atleast one antibody of the present invention. The method may additionallycomprise the administration of a second therapeutic agent. The secondtherapeutic agent may be an anti-viral agent. The anti-viral agent maybe Abacavir, Aciclovir, Adefovir, Amantadine, Amprenavir, Ampligen,Aplaviroc, Arbidol, Atazanavir, Atripla, Boceprevir, Cidofovir,Combivir, Darunavir, Delavirdine, Didanosine, Docosanol, Edoxudine,Efavirenz, Emtricitabine, Enfuvirtide, Entecavir, Entry inhibitors,Famciclovir, Fomivirsen, Fosamprenavir, Foscarnet, Fosfonet, Fusioninhibitor, Ganciclovir, Ibacitabine, Imunovir, Idoxuridine, Imiquimod,Indinavir, Inosine, Integrase inhibitor, Interferon (e.g., type I, II orIII), Lamivudine, Lopinavir, Loviride, Maraviroc, Moroxydine,Methisazone, Nelfinavir, Nevirapine, Nexavir, Nucleoside analogues,Oseltamivir (Tamiflu), Peginterferon alfa-2a, Penciclovir, Peramivir,Pleconaril, Podophyllotoxin, Protease inhibitor, Raltegravir, Reversetranscriptase inhibitor, Ribavirin, Rimantadine, Ritonavir, Pyramidine,Saquinavir, Stavudine, Tenofovir, Tenofovir disoproxil, Tipranavir,Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir (Valtrex),Valganciclovir, Vicriviroc, Vidarabine, Viramidine, Vicriviroc,Zalcitabine, Zanamivir (Relenza) or Zidovudine or a combination thereof.

The present invention also relates to methods of immunizing or reducingthe effect of an HIV infection or an HIV related disease which maycomprise identifying a patient in need of such treatment andadministering to said patient a therapeutically effective amount of: afirst antibody of the present invention, or fragment thereof, specificfor a first epitope which binds to said first antibody and a secondantibody of the present invention, or fragment thereof, specific for asecond epitope which binds to said second antibody. In one embodiment,the first antibody may be VRC-PG-04 or VRC-PG-05. In another embodiment,the second antibody may be VRC-PG-04 or VRC-PG-05. In yet anotherembodiment, the second antibody may be VRC01, VRC02, VRC03, VRCCH30,VRCCH31, VRCCH32, VRCCH33 or VRCCH34.

Identifying a patient in need of treatment for an HIV infection or anHIV related disease is known to one of skill in the art. A patient inneed of treatment for an HIV infection or an HIV related disease mayalso be identified by detecting HIV. In particular, HIV may be detectedby an HIV test such as an antibody test (e.g., ELISA or western blot)for HIV and/or a nucleic acid test (e.g., RT-PCR). Generally, infectionwith HIV-1 is associated with a progressive decrease of the CD4⁺ T cellcount and an increase in the level of HIV in the blood. The stage ofinfection may be determined by measuring the patient's CD4⁻ T cell countand viral load.

The compositions of the invention may be injectable suspensions,solutions, sprays, lyophilized powders, syrups, elixirs and the like.Any suitable form of composition may be used. To prepare such acomposition, a nucleic acid or vector of the invention, having thedesired degree of purity, is mixed with one or more pharmaceuticallyacceptable carriers and/or excipients. The carriers and excipients mustbe “acceptable” in the sense of being compatible with the otheringredients of the composition. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include, but are not limited to, water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol, or combinations thereof,buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

An immunogenic or immunological composition can also be formulated inthe form of an oil-in-water emulsion. The oil-in-water emulsion can bebased, for example, on light liquid paraffin oil (European Pharmacopeatype); isoprenoid oil such as squalane, squalene, EICOSANE™ ortetratetracontane; oil resulting from the oligomerization of alkene(s),e.g., isobutene or decene; esters of acids or of alcohols containing alinear alkyl group, such as plant oils, ethyl oleate, propylene glycoldi(caprylate/caprate), glyceryl tri(caprylate/caprate) or propyleneglycol dioleate; esters of branched fatty acids or alcohols, e.g.,isostearic acid esters. The oil advantageously is used in combinationwith emulsifiers to form the emulsion. The emulsifiers can be nonionicsurfactants, such as esters of sorbitan, mannide (e.g., anhydromannitololeate), glycerol, polyglycerol, propylene glycol, and oleic,isostearic, ricinoleic, or hydroxystearic acid, which are optionallyethoxylated, and polyoxypropylenepolyoxyethylene copolymer blocks, suchas the Pluronic® products, e.g., L121. The adjuvant can be a mixture ofemulsifier(s), micelle-forming agent, and oil such as that which iscommercially available under the name Provax® (IDEC Pharmaceuticals, SanDiego, Calif.).

The immunogenic compositions of the invention can contain additionalsubstances, such as wetting or emulsifying agents, buffering agents, oradjuvants to enhance the effectiveness of the vaccines (Remington'sPharmaceutical Sciences, 18th edition, Mack Publishing Company, (ed.)1980).

Adjuvants may also be included. Adjuvants include, but are not limitedto, mineral salts (e.g., AlK(SO4)2, AlNa(SO4)2, AlNH(SO4)2, silica,alum, Al(OH)₃, Ca₃(PO₄)₂, kaolin, or carbon), polynucleotides with orwithout immune stimulating complexes (ISCOMs) (e.g., CpGoligonucleotides, such as those described in Chuang, T. H. et al, (2002)J. Leuk. Biol. 71(3): 538-44; Ahmad-Nejad, P. et al (2002) Eur. J.Immunol. 32(7): 1958-68; poly IC or poly AU acids, polyarginine with orwithout CpG (also known in the art as IC31; see Schellack, C. et al(2003) Proceedings of the 34th Annual Meeting of the German Society ofImmunology; Lingnau, K. et al (2002) Vaccine 20(29-30): 3498-508),JuvaVax™ (U.S. Pat. No. 6,693,086), certain natural substances (e.g.,wax D from Mycobacterium tuberculosis, substances found inCornyebacterium parvum, Bordetella pertussis, or members of the genusBrucella), flagellin (Toll-like receptor 5 ligand; see McSorley, S. J.et al (2002) J. Immunol. 169(7): 3914-9), saponins such as QS21, QS17,and QS7 (U.S. Pat. Nos. 5,057,540; 5,650,398; 6,524,584; 6,645,495),monophosphoryl lipid A, in particular, 3-de-O-acylated monophosphoryllipid A (3D-MPL), imiquimod (also known in the art as IQM andcommercially available as Aldara®; U.S. Pat. Nos. 4,689,338; 5,238,944;Zuber, A. K. et al (2004) 22(13-14): 1791-8), and the CCR5 inhibitorCMPD167 (see Veazey, R. S. et al (2003) J. Exp. Med. 198: 1551-1562).

Aluminum hydroxide or phosphate (alum) are commonly used at 0.05 to 0.1%solution in phosphate buffered saline. Other adjuvants that can be used,especially with DNA vaccines, are cholera toxin, especiallyCTA1-DD/ISCOMs (see Mowat, A. M. et al (2001) J. Immunol. 167(6):3398-405), polyphosphazenes (Allcock, H. R. (1998) App. OrganometallicChem. 12(10-11): 659-666; Payne, L. G. et al (1995) Pharm. Biotechnol.6: 473-93), cytokines such as, but not limited to, IL-2, IL-4, GM-CSF,IL-12, IL-15 IGF-1, IFN-α, IFN-β, and IFN-γ (Boyer et al., (2002) J.Liposome Res. 121:137-142; WO01/095919), immunoregulatory proteins suchas CD40L (ADX40; see, for example, WO03/063899), and the CD1a ligand ofnatural killer cells (also known as CRONY or α-galactosyl ceramide; seeGreen, T. D. et al, (2003) J. Virol. 77(3): 2046-2055),immunostimulatory fusion proteins such as IL-2 fused to the Fc fragmentof immunoglobulins (Barouch et al., Science 290:486-492, 2000) andco-stimulatory molecules B7.1 and B7.2 (Boyer), all of which can beadministered either as proteins or in the form of DNA, on the sameexpression vectors as those encoding the antigens of the invention or onseparate expression vectors.

In an advantageous embodiment, the adjuvants may be lecithin is combinedwith an acrylic polymer (Adjuplex-LAP), lecithin coated oil droplets inan oil-in-water emulsion (Adjuplex-LE) or lecithin and acrylic polymerin an oil-in-water emulsion (Adjuplex-LAO) (Advanced BioAdjuvants(ABA)).

The immunogenic compositions can be designed to introduce the nucleicacids or expression vectors to a desired site of action and release itat an appropriate and controllable rate. Methods of preparingcontrolled-release formulations are known in the art. For example,controlled release preparations can be produced by the use of polymersto complex or absorb the immunogen and/or immunogenic composition. Acontrolled-release formulation can be prepared using appropriatemacromolecules (for example, polyesters, polyamino acids, polyvinyl,pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) known to provide thedesired controlled release characteristics or release profile. Anotherpossible method to control the duration of action by acontrolled-release preparation is to incorporate the active ingredientsinto particles of a polymeric material such as, for example, polyesters,polyamino acids, hydrogels, polylactic acid, polyglycolic acid,copolymers of these acids, or ethylene vinylacetate copolymers.Alternatively, instead of incorporating these active ingredients intopolymeric particles, it is possible to entrap these materials intomicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacrylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed in NewTrends and Developments in Vaccines, Voller et al. (eds.), UniversityPark Press, Baltimore, Md., 1978 and Remington's PharmaceuticalSciences, 16th edition.

Suitable dosages of the nucleic acids and expression vectors of theinvention in the immunogenic composition of the invention can be readilydetermined by those of skill in the art. For example, the dosage of theantibodies can vary depending on the route of administration and thesize of the subject. Suitable doses can be determined by those of skillin the art, for example by measuring the immune response of a subject,such as a laboratory animal, using conventional immunologicaltechniques, and adjusting the dosages as appropriate. Such techniquesfor measuring the immune response of the subject include but are notlimited to, chromium release assays, tetramer binding assays, IFN-γELISPOT assays, IL-2 ELISPOT assays, intracellular cytokine assays, andother immunological detection assays, e.g., as detailed in the text“Antibodies: A Laboratory Manual” by Ed Harlow and David Lane.

When provided prophylactically, the immunogenic compositions of theinvention are ideally administered to a subject in advance of HIVinfection, or evidence of HIV infection, or in advance of any symptomdue to AIDS, especially in high-risk subjects. The prophylacticadministration of the immunogenic compositions can serve to provideprotective immunity of a subject against HIV-1 infection or to preventor attenuate the progression of AIDS in a subject already infected withHIV-1. When provided therapeutically, the immunogenic compositions canserve to ameliorate and treat AIDS symptoms and are advantageously usedas soon after infection as possible, preferably before appearance of anysymptoms of AIDS but may also be used at (or after) the onset of thedisease symptoms.

The immunogenic compositions can be administered using any suitabledelivery method including, but not limited to, intramuscular,intravenous, intradermal, mucosal, and topical delivery. Such techniquesare well known to those of skill in the art. More specific examples ofdelivery methods are intramuscular injection, intradermal injection, andsubcutaneous injection. However, delivery need not be limited toinjection methods. Further, delivery of DNA to animal tissue has beenachieved by cationic liposomes (Watanabe et al., (1994) Mol. Reprod.Dev. 38:268-274; and WO 96/20013), direct injection of naked DNA intoanimal muscle tissue (Robinson et al., (1993) Vaccine 11:957-960;Hoffman et al., (1994) Vaccine 12: 1529-1533; Xiang et al., (1994)Virology 199: 132-140; Webster et al., (1994) Vaccine 12: 1495-1498;Davis et al., (1994) Vaccine 12: 1503-1509; and Davis et al., (1993)Hum. Mol. Gen. 2: 1847-1851), or intradermal injection of DNA using“gene gun” technology (Johnston et al., (1994) Meth. Cell Biol.43:353-365). Alternatively, delivery routes can be oral, intranasal orby any other suitable route. Delivery also be accomplished via a mucosalsurface such as the anal, vaginal or oral mucosa.

Immunization schedules (or regimens) are well known for animals(including humans) and can be readily determined for the particularsubject and immunogenic composition. Hence, the immunogens can beadministered one or more times to the subject. Preferably, there is aset time interval between separate administrations of the immunogeniccomposition. While this interval varies for every subject, typically itranges from 10 days to several weeks, and is often 2, 4, 6 or 8 weeks.For humans, the interval is typically from 2 to 6 weeks. Theimmunization regimes typically have from 1 to 6 administrations of theimmunogenic composition, but may have as few as one or two or four. Themethods of inducing an immune response can also include administrationof an adjuvant with the immunogens. In some instances, annual, biannualor other long interval (5-10 years) booster immunization can supplementthe initial immunization protocol.

The present methods also include a variety of prime-boost regimens, forexample DNA prime-Adenovirus boost regimens. In these methods, one ormore priming immunizations are followed by one or more boostingimmunizations. The actual immunogenic composition can be the same ordifferent for each immunization and the type of immunogenic composition(e.g., containing protein or expression vector), the route, andformulation of the immunogens can also be varied. For example, if anexpression vector is used for the priming and boosting steps, it caneither be of the same or different type (e.g., DNA or bacterial or viralexpression vector). One useful prime-boost regimen provides for twopriming immunizations, four weeks apart, followed by two boostingimmunizations at 4 and 8 weeks after the last priming immunization. Itshould also be readily apparent to one of skill in the art that thereare several permutations and combinations that are encompassed using theDNA, bacterial and viral expression vectors of the invention to providepriming and boosting regimens.

A specific embodiment of the invention provides methods of inducing animmune response against HIV in a subject by administering an immunogeniccomposition of the invention, preferably which may comprise anadenovirus vector containing DNA encoding one or more of the antibodiesof the invention, one or more times to a subject wherein the epitopesare expressed at a level sufficient to induce a specific immune responsein the subject. Such immunizations can be repeated multiple times attime intervals of at least 2, 4 or 6 weeks (or more) in accordance witha desired immunization regime.

The immunogenic compositions of the invention can be administered alone,or can be co-administered, or sequentially administered, with other HIVimmunogens and/or HIV immunogenic compositions, e.g., with “other”immunological, antigenic or vaccine or therapeutic compositions therebyproviding multivalent or “cocktail” or combination compositions of theinvention and methods of employing them. Again, the ingredients andmanner (sequential or co-administration) of administration, as well asdosages can be determined taking into consideration such factors as theage, sex, weight, species and condition of the particular subject, andthe route of administration.

The immunogenic compositions of the invention can be administered alone,or can be co-administered, or sequentially administered, with othertherapeutic agents, thereby providing multivalent or “cocktail” orcombination compositions of the invention and methods of employing them.The therapeutic agent can be an antiviral agent. Useful antiviral agentsinclude, but are not limited to, nucleoside analogs, such as zidovudine,acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, andribavirin, as well as foscarnet, amantadine, rimantadine, saquinavir,indinavir, ritonavir, and the alpha-interferons. Again, the ingredientsand manner (sequential or co-administration) of administration, as wellas dosages can be determined taking into consideration such factors asthe age, sex, weight, species and condition of the particular subject,and the route of administration.

When used in combination, the other HIV immunogens can be administeredat the same time or at different times as part of an overallimmunization regime, e.g., as part of a prime-boost regimen or otherimmunization protocol. In an advantageous embodiment, the other HIVimmunogen is env, preferably the HIV env trimer.

Many other HIV immunogens are known in the art, one such preferredimmunogen is HIVA (described in WO 01/47955), which can be administeredas a protein, on a plasmid (e.g., pTHr.HIVA) or in a viral vector (e.g.,MVA.HIVA). Another such HIV immunogen is RENTA (described inPCT/US2004/037699), which can also be administered as a protein, on aplasmid (e.g., pTHr.RENTA) or in a viral vector (e.g., MVA.RENTA).

For example, one method of inducing an immune response against HIV in ahuman subject may comprise administering at least one priming dose of anHIV immunogen and at least one boosting dose of an HIV immunogen,wherein the immunogen in each dose can be the same or different,provided that at least one of the immunogens is an epitope of thepresent invention, a nucleic acid encoding an epitope of the inventionor an expression vector, preferably a VSV vector, encoding an epitope ofthe invention, and wherein the immunogens are administered in an amountor expressed at a level sufficient to induce an HIV-specific immuneresponse in the subject. The HIV-specific immune response can include anHIV-specific T-cell immune response or an HIV-specific B-cell immuneresponse. Such immunizations can be done at intervals, preferably of atleast 2-6 or more weeks.

The invention will now be further described by way of the followingnon-limiting examples.

EXAMPLES Example 1 Use of RSC3 and RSC3 Mutant Probes to Isolate Two NewNeutralizing Monoclonal Antibodies from IAVI Protocol G Donor PBMC

The Protocol G serum and PBMC analysis was performed as follows. A setof 12 broadly neutralizing sera was first analyzed by resurfacesstabilized core (RSC3) ELISA for evidence of CD4 binding site (CD4bs)directed antibodies. Positive sera was further analyzed in competitionneutralization assay using RSC3 to block serum neutralization. Thisdemonstrated that sera have neutralizing antibodies (NAbs) to the CD4bs.Four out of twelve ( 4/12) sera had evidence of CD4bs directedneutralization; one was chosen for mAb isolation (VRC code was IAVI#2,corresponding to IAVI sample #27-374).

Monoclonal antibody isolation method was performed as follows. Themethod used was essentially the same as described in Wu et al, Science329; 856 (2010). PBMC were incubated with RSC3 and ΔRSC3 to find B-cellsreactive with RSC3 and not ΔRSC3; Single B-cells sorted into 96 wellplates. Antibody heavy and light chain variable region genes amplifiedby PCR and cloned into expression vectors (one for heavy and one forlight) and full IgG expressed.

FIG. 1 depicts protein competition that shows that serum 27-374 hasCD4bs directed neutralizing antibodies. Neutralization by IAVI serum27-374 against a panel of viruses shown on x-axis. Bars show the %reduction in neutralization for each virus when the RSC3 protein isadded as a competitor. The ΔRSC3 is a knock out mutant. Data show that˜50% of the serum neutralization against each virus could be blocked byRSC3—indicating the presence of CD4bs directed neutralizing antibodies.

FIG. 2 depicts single cell sort for RSC3 reactive B-cells, in particulara sort profile from donor PBMC: 27-374. Schema for isolating thespecific B-cells that produced VRC-PG-04 and VRC-PG-05. Donor PBMC wereincubated with wild type form of cloak-2a protein and the Δ371 mutantprotein. The RSC3 protein is highly specific for anti-CD4-binding siteantibodies; the mutant does not bind such antibodies. Both proteins arebiotin labeled and then labeled with different color fluorochromes. Flowcytometry was performed to isolate memory B-cells and to furtheridentify B-cells that bind RSC3, and not the A 371 mutant (0.075% ofmemory B-cells). These B-cells were deposited as a single cell per well,into 96-well plates.

FIG. 3 depicts ELISA Binding of VRC-PG-04 and VRC-PG-05 to severalmutant versions of YU2 gp120 and to the resurfaced stabilized core(RSC3) proteins and its knock out mutant ΔRSC3. ELISA data showed thatVRC-PG04 binds well to gp120 and to RSC3, with reduced binding to theCD4bs knock out mutants of those two proteins. Therefore, it is likelydirected to the CD4bs of gp120. VRC-PG-05 bound better to RSC3 than togp120, thus its epitope appears to be distinct from VRC-PG-04.

FIG. 4 depicts binding of VRC-PG-04 and VRC-PG-05 to YU2 gp120-basedproteins, HXB2 new core 8b and Du156 gp120 WT by ELISA. VRC-PG-05 bindswell to the HXB2 new core (right graph) indicating that its epitope lieswithin the core of gp120, but is different from. VRC-PG-04 which bindsstrongly to the clade C Du156 gp120 whereas VRC-PG-05 binds only weakly.

FIG. 5 depicts competition ELISA demonstrating that VRC-PG-04 isdirected against CD4bs. Data are competition ELISAs on either YU2 gp120(left graphs) or HxB2 gp120 (right graphs). Top graphs show binding ofCD4-Ig in the presence of several other IgG mAbs. Bottom graphs showbinding of 17b in the presence of other mAbs. The previously publishedVRC01 and VRC03 mAbs are included (Wu et al, Science 329; 856 (2010)).VRC-PG-04 cross competes with CD4-Ig and with VRC01—showing it is likelydirected to the CD4bs of gp120. Bottom graphs show that VRC-PG-04 doesnot potentiate 17b binding on YU2 gp120, though it mildly potentiates17b binding to HxB2 gp120.

FIG. 6 depicts competition ELISA demonstrating that VRC-PG-04, but notVRC-PG-05, is cross competed by CD4bs antibodies ELISAs done with RSC3on plate. Competition ELISA with RSC3 protein on plate. Top graph showsthat VRC-PG-04 is cross blocked by other mAbs directed to the CD4bs. Incontrast, VRC-PG-05 is not cross blocked by mAbs directed to the CD4bs,indicating it is directed against a different epitope on the coreprotein of gp120. The previously published VRC01, VRC02 and VRC03 mAbs(Wu et al, Science 329; 856 (2010)) are included for comparison.

FIG. 7 depicts competition ELISA using clade B AC10.29 gp1120 on plateand that VRC-PG-05 does not cross-block CD4bs mAbs. Competition ELISAwith strain AC10.29 gp120 protein on plate. The top graph shows thatCD4-Ig is cross blocked by itself, but not by VRC-PG-05. The bottomgraph shows the VRC-PG-05 is cross blocked by itself, but not by any ofthe other mAbs tested. Thus, VRC-PG-05 binds to a unique epitope ongp120. The previously published VRC01, VRC02 and VRC03 mAbs (Wu et al,Science 329; 856 (2010)) are included for comparison.

Table 1 depicts a gene family analysis of VRC-PG-04 and VRC-PG-05compared to mAbs b12, VRC01, VRC02 and VRC03. The previously publishedVRC01, VRC02 and VRC03 mAbs (Wu et al, Science 329; 856 (2010)) areincluded for comparison. The table shows the inferred germline V, D andJ genes for the heavy chain and the V and J genes for the kappa lightchain. VRC-PG-04 and VRC-PG-05 are heavily mutated from germline.

TABLE 1 Repertoire and mutation analysis of VRC mAbs. Heavy Chain VHmutation CDR3 length frequency IGHV IGHD IGHJ (amino acids)(nucleotides) VRC01 1-02*02 3-16*01 1*01 14 91/288 (32%) (or *02) VRC021-02*02 3-16*01 1*01 14 92/288 (32%) (or *02) VRC03 1-02*02 IGHD3 1*0116 85/288 (30%) family b12 1-03*01 3-10*02 6*03 20 39/288 (13%)VRC-PG-04 1-02*02 2-8*02 2*01 16 84/288 (29%) VRC-PG-05 3-7*01 3-3*013*02 19 27/288 (9%) Light Chain VL mutation CDR3 length frequency IGKVIGKJ (amino acids) (nucleotides) VRC01 3-11*01 2*01 5 45/264 (17%) VRC023-11*01 2*01 5 49/264 (19%) VRC03 3-20*01 2*01 5 53/267 (20%) b123-20*01 2*01 9 35/267 (13%) VRC-PG-04 3-20*01 5*01 5 51/267 (19%)VRC-PG-05 4-1*01 2*03 8 21/297 (7%)

FIG. 8 depicts sequence alignments of VRC-PG-04 and VRC-PG-05 comparedto their inferred germline sequence. The previously published VRC01,VRC02 and VRC03 mAbs (Wu et al, Science 329; 856 (2010)) are includedfor comparison.

Example 2 VRC-PG-04 Isolation

A novel monoclonal antibody, VRC-PG-04, was isolated from an HIV+ donorusing an antigentically resurfaced, stabilized gp120 glycoprotein probethat was specific for the CD4 binding site. VRC-PG-04 competed withCD4-IgG for binding to gp120, demonstrating that VRC-PG-04 is directedagainst the CD4bs. This antibody is potent and broadly neutralizing,having a mean IC50 of 0.172 μg/ml and being able to neutralize a widerange of pseudovirus entry into TZM-bl cells, including pseudovirus fromclade A—87% (n=24), B—96% (n=26), and C—79% (n=34).

To further characterize the Ab, neutralization of entry assays werecarried out using a panel of JRCSF gp120 single alanine mutantsincorporated into pseudovirus. Applicants found that neutralization ofentry by VRC-PG-04 was knocked out (as defined by having less than 10%neutralization potency compared to wildtype) by a single mutation in aaD279 (see Neutralization Potency chart). Applicants next determinedwhich residues were important for Ab binding to gp120. Applicants foundbinding of VRC-PG-04 to several gp120 glycoproteins isolated frompseudovirus bearing mutant JRSCF gp120s was drastically decreased asmeasured by ELISA. In particular, these residues were found in V1N2 stem(N197), C2 (N276, D279, N280), C3 (1371), and C4 (T450, R456). InApplicants' preliminary screen, there seem to be less residues involvedin knocking out binding (as defined by having less than 10% apparentaffinity to gp120 compared to wildtype) of VRC-PG-04 to gp120 whencompared to the number of residues involved in knocking out binding togp120 of b12 or CD4 (see Table 2, binding chart).

TABLE 2 Binding chart Apparent Affinity Domain Mutant pub b12 pub CD42G12 VRC- PG-04 E87A 7 114 53 M95A 75 59 41 C1 K97A 134 50 98 E102A 6347 50 W112A 2 67 76 D113A 1 9 169 70 V120A 79 K121A 4 3 130 C1 L122A 2762 96 (V1/V2-stem) T123A 233 42 113 L125A 383 23 93 V127A 62 26 22 N156A72 N160K 390 T162A 83 I165A 20 57 139 R166A 52 44 97 D167A 97 K168A 34K171A 118 26 78 E172A 143 93 74 V2 F176A 2 19 400 104 Y177A 87 L179A 138D180A 1 31 52 V182A 54 I184A 0.6 27 27 D185A 6 62 55 T190A 21 187 42K194S 22 N197A 25 3 318 10 C2 T198A 0.1 42 49 (V1/V2-stem) S199A 123 478 T202A 220 62 58 K207A 2 5 100 31 F210A 77 37 85 I213A 20 31 114 28R252A 17 47 111 S256A 13 47 108 37 T257A 28 21 133 51 C2 N262A 6 5 17R273A 0.9 40 78 N276A 225 27 169 1 D279A 27 33 0 N280A 75 44 0 K282A 3278 23 T283A 55 53 85 PGV04 N295A 34 T297S 115 V3 (base) P299A 115 N301A43 N302A 40 V3 (stem) R304A 38 K305A 51 S306A 93 VRC- PG-04 R456A 6 53 8D457A 5 2 G458A 300 2 G459A 54 37 61 N461A 4 160 285 E462A 21 32 178S463A 50 53 324 V5 I467A 63 3 R469A 1.4 8 P470A 1.3 13 100 G471A 37 89137 G472A 22 2 100 G473A 998 2 D474A 91 22 C5 M475A 130 64 R476A 25 40133 81 D477A 2 187 44 W479A 1.3 19 40 95 R480A 37 73 Delta V1 35 44 133V1-V3 Delta V1/2 30 12 114 Delta V3 30 41 apparent affinity relative toWT = (apparent affinity for WT/apparent affinity for mutant) * 100 note:graph adapted from Pantophlet et al., 2003.

TABLE 3 Neutralization Potency chart. The previously published VRC01 andVRC03 mAbs (Wu et al., Science 329; 856 (2010)) are included forcomparison. Neutralization potency gp120 relative to WT (%) domainmutation CD4 b12 VRC01 VRC03 VRC-PG-04 C1 E87A 46 29 63 106 118 M95A 1748 56 79 107 K97A 184 117 62 108 130 E102A 60 83 98 112 146 W112A 100119 78 111 118 C1 (V1/V2 V120A 161 80 60 101 84 stem) K121A 28 30 24 6978 L122A 474 96 66 51 106 T123A 121 L125A 9532 1549 222 29 89 N156A 234568 30 454 N160K 99 173 296 173 176 V2 T162A 318 107 190 21 177 I165A 43441 42 10 61 R166A 22 41 42 31 51 D167A 702 121 99 23 81 K168A 106 80 5050 67 K171A 52 58 60 132 149 E172A 259 55 161 34 59 Y177A 2416 89 1 135L179A 1526 19 13 25 88 V182A 128 45 60 76 68 I184A 806 172 129 94 155D185A 96 4 34 88 120 T190A 8 165 119 120 82 C2 (V1/V2 K194S 1502 41001538 906 236 stem) T198A 218 5 40 22 95 S199A 2903 5973 724 145 707T202A 3831 569 30 3 61 C2 F210A 845 384 163 62 101 I213A 102 178 174 186185 R252A 21 98 68 ND 86 S256A 112 83 73 136 152 T257A 5 107 253 86 148N262A 63 443 223 12 105 R273A 132 109 48 72 120 N276A 29 134 341 536 18D279A 2 92 3 0.5 0.4 K282A 5 78 363 99 81 T283A 5 717 346 99 225 V3(base) N295A 46 85 56 97 97 T297S 17 47 50 137 179 P299A 4882 80 0.50115 N301A 8163 1396 239 22 507 V3 (stem) N302A 28 75 43 110 120 R304A4497 125 32 0.5 43 K305A 3466 70 0.5 54 PGV04 V3 (tip) S306A 246 94 76 7130 I307A 7399 119 1 20 H308A 505 76 66 4 99 I309A 3493 82 1 31 P313A 313 130 55 111 R315A 393 67 45 14 92 F317A 4709 116 1 37 Y318A 4846 109 127 T319A 316 54 56 34 102 T320A 1240 56 2 64 V3 (base) E322A 293 76 11026 83 D325A 444 154 53 26 112 N332A 118 143 119 73 120 C3 Q337A 20 56 4353 172 K343A 34 65 45 92 117 R350A 21 76 60 38 106 S365A 32 70 114 85155 P369A 26 30 72 86 113 V372A 12 12 74 20 125 M373A 37 124 90 20 135Y384A 69 N386A 50 392 212 79 120 T388A 110 482 196 15 160 V4 N392Q 29 6344 62 73 W395A 95 C4 R419A 596 30 120 17 111 I420A 1015 344 16 1 21K421A 1457 64 1 68 Q422A 5369 657 47 14 98 I423A 3412 19 1 24 I424A 230126 1 142 N425A 22 98 61 34 148 V430A 4336 36 1 765 K432A 147 61 55 30101 Y435A 3407 2034 31 1 73 I439A 2854 656 162 1508 339 T450A 31 126 84115 135 T455A 22 27 79 ND 142 R456A 55 106 63 1 259 G459A 722 69 149 1239 V5 N461A 97 90 363 496 321 E462A 38 49 46 106 116 S463A 54 109 536296 461 G471A 10 70 25 96 92 C5 D474A 11 109 32 8 102 M475A 61 59 68 55140 R476A 5 129 200 26 141 D477A 10 158 68 10 107 W479A 70 508 160 51138 R480A 53 150 200 51 113 blue = 1-10% green = 11-40% yellow =250-10,000% red >10,000%

Example 3 Neutralization Data of VRC-PG-04 and VRC-PG-05

Neutralization performed with Env-pseudoviruses using TZM-bl targetcells. The values in Table 4 represent mAb concentration required toachieve 50% (Table 4A) or 80% (Table 4B) neutralization.

All monoclonal antibodies tested are IgG. CD4-Ig is chimeric 5 bivalentIgG-CD4 construct. VRC01, VRC02 and VRC03, b12, PG9 and PG16 werepreviously published and are shown for comparison.

Note: Breadth and potency calculations excluded Tier 1 viruses fromclades B and C. Potency was calculated using viruses that have an IC50or IC80 value within the tested range.

VRC-PG- VRC-PG- VRC01 VRC02 VRC03 04 05 b12 CD4-Ig PG9 PG16 Tier 1 HXB20.034 0.042 0.048 0.025 13 0.007 0.005 1.62 >50 clade B MN.3 0.022 0.0240.027 >50 >50 0.003 0.006 >50 >50 (n = 7) SF162 0.139 0.112 0.033 0.0246.84 0.070 0.153 >50 >50 ADA 0.379 0.391 0.113 0.179 4.33 0.131 0.0510.128 0.012 BaL.01 0.055 0.053 20.1 0.034 0.258 0.093 0.030 0.033 0.993BaL.26 0.048 0.046 10.4 0.148 0.253 0.051 0.047 0.019 0.131 SS1196.10.170 0.132 0.048 0.189 >50 0.840 0.571 0.074 0.020 Tier 1 MW965.260.056 0.057 >50 0.032 5.07 0.19 0.039 2.17 0.476 clade C Clade A RW020.20.224 0.123 >50 0.165 >50 10.1 11.7 0.052 0.037 (n = 24) UG037.8 0.0790.082 12.1 0.109 >50 >50 0.134 0.020 0.010 DJ263.8 0.080 0.055 >500.803 >50 0.812 0.088 0.218 8.21 KER2018.11 0.652 0.516 0.3891.08 >50 >50 3.34 0.010 0.004 Q259.w6 0.170 0.147 0.055 0.028 7.03 >500.708 1.17 2.58 Q769.h5 0.084 0.047 0.034 0.024 0.010 >50 1.28 0.0090.009 Q168.a2 0.115 0.092 3.38 0.032 9.92 >50 11.6 0.045 0.019 Q23.170.085 0.071 0.065 0.084 >50 >50 12.7 0.005 0.002 Q259.17 0.066 0.0470.027 >50 >50 >50 7.38 0.041 0.030 Q461.e2 0.492 0.463 >50 0.23739.3 >50 25.4 1.47 2.74 Q842.d12 0.030 0.025 >50 0.017 5.12 >50 >500.019 0.009 BB201.B42 0.223 0.164 7.93 0.081 23.6 0.358 14.1 0.011 0.002MB201.A1 0.165 0.089 >50 0.049 12.9 >50 >50 0.054 0.021 MB201.B10 0.1320.095 >50 0.042 9.24 >50 >50 0.052 0.020 BB539.2B13 0.069 0.059 8.580.398 >50 0.624 1.700 0.063 0.012 MB539.2D1 0.060 0.046 17 0.499 >500.476 12.1 0.035 0.009 MB539.2B7 0.531 0.383 >50 0.462 >50 11.6 7.910.094 0.032 BI369.9A 0.142 0.101 >50 0.039 9.35 28.9 10.5 0.023 0.010MI369.A5 0.107 0.086 >50 0.046 16.4 4.05 4.35 0.035 0.012 BS208.B1 0.0190.014 0.297 0.014 5.77 0.042 0.246 0.016 0.003 MS208.A1 0.101 0.074 >500.055 21.6 0.201 7.94 0.032 0.008 MS208.A3 0.050 0.037 >50 0.022 8.290.505 3.51 0.025 0.006 KER2008.12 0.379 0.265 0.403 0.236 >50 >50 0.6490.017 0.008 KNH1209.18 0.087 0.095 45 0.058 13.6 0.227 5.95 0.167 0.283Tier 2 JRCSF.JB 0.093 0.099 0.093 0.034 35 0.096 0.186 0.002 0.001 cladeB JRFL 0.031 0.024 0.009 0.063 3.09 0.022 0.247 >50 >50 (n = 26) YU20.126 0.115 0.037 0.084 35 2.18 0.102 1.73 0.114 89.6 0.511 0.444 0.1870.061 18.8 0.14 0.242 >50 >50 6101.10 0.111 0.135 0.094 0.090 6.77 >502.7 >50 >50 7165 16.3 >50 >50 >50 >50 >50 2.85 >50 0.426 6535 0.5390.733 0.438 0.687 >50 0.429 2.49 0.222 >50 QH0692.42 1.5 1.33 0.9541.34 >50 0.97 0.603 >50 >50 SC422661.8 0.076 0.084 0.036 0.038 12.8 0.445.19 0.325 19.1 PVC.4 0.216 0.168 0.328 0.235 >50 >50 20.1 8.7 12.0TRO.11 0.207 0.208 0.055 0.131 >50 >50 >50 >50 0.136 AC10.0.29 2.22.48 >50 17.9 0.017 1.8 10.7 0.012 0.007 RHPA4259.7 0.060 0.086 1.130.038 9.6 0.12 1.09 10 0.334 THRO4156.18 2.25 3.43 >50 >50 >50 1.210.509 13.2 0.498 REJO4541.67 0.062 0.056 0.059 0.019 1.64 5.92 1.220.001 0.004 TRJO4551.58 0.083 0.115 0.043 0.069 >50 >50 22.1 1.85 2.7WITO4160.33 0.148 0.115 >50 0.080 >50 8.54 2.17 0.005 0.002 CAAN5342.A20.824 0.899 8.32 1.13 0.005 >50 >50 14.4 25.0BL01.DG >50 >50 >50 >50 >50 1.650 0.100 >50 >50 BR07.DG 1.24 0.948 3.380.789 >50 0.096 0.046 >50 >50 HT593.1 0.334 0.542 0.235 0.177 0.3890.117 0.323 0.214 0.056 R2 0.198 0.242 0.035 0.291 >50 1.1700.016 >50 >50 BG1168.01 0.276 0.458 >50 0.509 >50 >50 13.4 >50 >50QH0515.01 0.386 0.470 0.187 0.115 >50 0.300 1.83 >50 >50 5768 0.1660.275 0.382 0.042 >50 0.249 0.756 0.031 0.008 3988 0.220 0.243 2.460.295 9.70 0.378 49.4 0.016 0.005 Tier 2 Du123.6 18.2 16.1 >50 >50 >501.82 0.142 0.047 0.016 clade C Du151.2 3.16 4.83 34.6 0.059 0.128 3.791.36 0.012 0.004 (n = 34) Du156.12 0.089 0.091 >50 0.034 7.58 0.656 14.50.035 0.002 Du172.17 >50 >50 >50 0.314 28.5 0.300 0.26 0.240 0.023Du422.1 >50 >50 >50 >50 3.7 0.464 11.5 0.178 0.042 ZM197M.PB7 0.36 0.4082.13 1.14 >50 >50 28.3 0.287 0.765 ZM214M.PL15 0.44 0.75 18.8 0.249 >5013.6 26.6 >50 >50 ZM233M.PB6 1.99 1.03 >50 7.67 >50 >50 3.36 0.001 0.001ZM249M.PL1 0.048 0.062 8.59 0.051 12.7 3.81 11.1 0.023 0.007 ZM53M.PB121.31 1.4 10.3 1.51 0.145 32.6 8.58 0.092 0.009 ZM109F.PB4 0.1280.127 >50 0.047 0.151 >50 0.028 0.235 9.8 ZM135M.PL10a 0.346 0.14 >5041 >50 >50 0.296 >50 >50 CAP45.2.00.G3 2.29 5.68 >50 >50 >50 0.37 2.110.003 0.002 CAP210.2.00.E8 >50 >50 >50 >50 >50 27 1.48 0.08 0.021CAP244.2.00.D3 0.428 0.688 47.1 0.301 >50 >50 2.56 0.082 0.014 ZA012.290.305 0.176 9.21 0.130 15.2 >50 5.39 4.59 0.414 BR025.9 0.115 0.208 >502.77 >50 >50 0.064 0.018 0.004 TV1.29 >50 >50 >50 >50 >50 >50 0.4050.007 0.005 ZM215.8 0.095 0.149 >50 0.075 41.9 >50 1.16 0.025 >50ZM106.9 0.489 0.378 0.150 0.206 38.4 >50 5.39 4.59 0.414 ZM55.28a 0.3400.326 >50 0.390 >50 >50 >50 4.60 >50 ZM53.21 1.16 1.25 3.46 1.15 0.1219.54 1.56 0.019 0.003 ZM55.4a 0.450 0.411 >50 0.457 >50 32.6 2.7 4.190308 ZM106.10 0.566 0.341 0.154 0.125 48.4 >50 41.4 0.097 0.042 ZM109.320.091 0.086 >50 0.055 0.151 >50 7.69 0.099 30 ZM135.8a 0.3740.533 >50 >50 >50 >50 13.7 >50 >50 ZM146.7 0.333 0.396 1.04 0.403 >50 184.21 0.181 0.32 ZM176.66 0.055 0.036 0.033 0.140 >50 >50 0.212 0.0110.002 ZM181.6 1.12 0.574 >50 11.6 >50 >50 4.9 0.005 0.001 SO18.18 0.0690.071 0.083 0.067 19.8 13.9 9.86 0.031 0.004 286.4 0.188 0.193 1.770.090 >50 0.701 7.3 0.084 0.012 288.4 0.992 0.749 0.342 0.390 >50 >500.459 0.610 0.083 TZA125.17 >50 >50 >50 >50 >50 >50 0.125 0.115 0.012TZBD.02 0.109 0.074 1.27 0.067 4.1 >50 0.895 0.211 0.013 Clade D UG024.20.156 0.103 >50 0.199 >50 >50 0.009 3.23 >50 57128 >50 >50 >50 >50 >500.169 0.112 0.136 0.076 clade E TH966.8 0.334 0.288 >50 0.068 4.90 >500.397 0.020 0.003 TH976.17 0.087 0.112 >50 0.046 7.48 >50 0.896 >50 >50M02138 0.348 0.450 >50 0.219 0.046 >50 0.297 0.189 0.020 Non-HIVSIVmac251.30 >50 >50 >50 >50 >50 >50 0.496 >50 >50MuLV >50 >50 >50 >50 >50 >50 >50 >50 >50 Breadth total n = 89 82/8981/89 49/89 77/89 44/89 47/89 82/89 74/89 72/89 (92%) (91%) (55%) (87%)(49%) (53%) (92%) (83%) (81%) clade A n = 24 24/24 24/24 13/24 23/2414/24 12/24 21/24 24/24 24/24 (100%)  (100%)  (54%) (96%) (58%) (50%)(88%) (100%)  (100%)  clade B n = 26 25/26 24/26 20/26 23/26 12/26 19/2624/26 15/26 16/26 (96%) (92%) (77%) (88%) (46%) (73%) (92%) (58%) (62%)clade C n = 34 29/34 29/34 16/34 27/34 15/34 15/34 32/34 31/34 29/34(85%) (85%) (47%) (79%) (44%) (44%) (94%) (91%) (85%) Potency* IC50median 0.203 0.168 0.389 0.125 8.765 0.701 2.170 0.053 0.014 IC50geometric mean 0.243 0.218 0.617 0.172 3.576 1.105 1.652 0.084 0.034

TABLE 4B IC₈₀ values (μg/ml) VRC- VRC- VRC01 VRC02 VRC03 PG-04 PG-05 b12CD4-Ig PG9 PG16 Tier 1 HXB2 0.111 0.143 0.165 0.113 >50 0.0160.010 >50 >50 clade B MN.3 0.082 0.071 0.073 >50 >50 0.007 0.018 >50 >50(n = 7) SF162 0.567 0.367 0.097 0.127 >50 0.270 0.539 >50 >50 ADA 1.491.25 0.315 0.700 47.5 0.656 0.209 5.18 0.060 BaL.01 0.214 0.178 >500.252 1.48 0.32 0.113 1.08 >50 BaL.26 0.176 0.151 >50 2.74 1.61 0.1550.145 0.112 >50 SS1196.1 0.593 0.411 0.096 0.649 >50 4.02 2.86 0.6950.179 Tier 1 MW965.26 0.167 0.173 >50 0.096 19.3 6.9 0.356 >50 >50 cladeC Clade A RW020.2 0.883 0.492 >50 0.581 >50 33.5 46.1 0.269 0.385 (n =24) UG037.8 0.313 0.263 >50 0.280 >50 >50 0.721 0.074 0.031 DJ263.80.553 0.424 >50 >50 >50 >50 0.557 2.62 >50 KER2018.11 2.3 1.9 1.253.62 >50 >50 15.9 0.033 0.011 Q259.w6 0.543 0.434 0.178 0.087 21.5 >502.83 >50 >50 Q769.h5 0.289 0.204 0.140 0.093 0.016 >50 5.74 0.033 0.067Q168.a2 0.362 0.310 27.8 0.112 29.4 >50 >50 0.173 0.078 Q23.17 0.2610.220 0.202 0.269 >50 >50 >50 0.012 0.005 Q259.17 0.233 0.1370.085 >50 >50 >50 19.6 0.166 0.488 Q461.e2 1.61 1.44 >500.758 >50 >50 >5.0 10.9 >50 Q842.d12 0.096 0.074 >50 0.046 14.5 >50 >500.070 0.031 BB201.B42 0.894 0.675 >50 0.298 >50 2.47 >50 0.030 0.008MB201.A1 0.634 0.310 >50 0.184 >50 >50 >50 0.193 0.108 MB201.B10 0.5380.374 >50 0.132 >50 >50 >50 0.218 0.125 BB539.2B13 0.286 0.224 >5028.6 >50 3.84 8.62 0.232 0.046 MB539.2D1 0.480 0.277 >50 9.61 >50 3.7229.6 0.122 0.030 MB539.2B7 1.44 1.06 >50 1.35 >50 >50 45.9 0.292 0.272BI369.9A 0.558 0.403 >50 0.132 >50 >50 38.5 0.086 0.045 MI369.A5 0.5880.464 >50 0.183 >50 30.9 22.3 0.191 0.099 BS208.B1 0.078 0.050 2.580.049 17.2 0.224 20.8 0.046 0.012 MS208.A1 0.462 0.353 >50 0.221 >501.12 40.9 0.164 0.095 MS208.A3 0.192 0.133 >50 0.092 28.6 4.65 >50 0.0860.020 KER2008.12 1.7 0.994 1.65 1.03 >50 >50 3.99 0.068 0.051 KNH1209.180.296 0.260 >50 0.201 >50 1.75 >50 19.1 >50 Tier 2 JRCSF.JB 0.544 0.4750.517 0.178 >50 0.874 1.65 0.007 0.006 clade B JRFL 0.093 0.075 0.0250.287 11.8 0.075 0.967 >50 >50 (n = 26) YU2 0.372 0.359 0.115 0.240 >507.79 0.314 13.4 1.27 89.6 2.32 1.46 0.589 0.221 >50 0.56 0.752 >50 >506101.10 0.315 0.384 0.184 0.247 29.1 >50 5.33 >50 >507165 >50 >50 >50 >50 >50 >50 33 >50 12.4 6535 2.74 3.76 2.4 4.2 >50 19.116.3 5.04 >50 QH0692.42 4.83 4.18 2.05 4.41 >50 2.67 2.63 >50 >50SC422661.8 0.265 0.267 0.105 0.147 >50 1.69 >50 >50 >50 PVO.4 1.19 1.031.68 1.44 >50 >50 >50 >50 >50 TRO.11 0.832 0.876 0.3420.744 >50 >50 >50 >50 >50 AC10.0.29 6.45 6.95 >50 >50 0.065 14.2 >500.073 0.023 RHPA4259.7 0.185 0.243 6.58 0.134 32.2 0.39 13.9 >50 3.49THRO4156.18 23 21.7 >50 >50 >50 4.64 2.5 >50 50 REJO4541.67 0.251 0.240.196 0.050 28.4 >50 11.5 0.01 0.030 TRJO4551.58 0.207 0.284 0.0980.183 >50 >50 >50 17.7 >50 WITO4160.33 0.412 0.35 >50 1.19 >50 41.4 13.20.009 0.006 CAAN5342.A2 2.77 3.13 47.6 5.83 0.012 >50 >50 >50 >50BL01.DG >50 >50 >50 >50 >50 >50 0.626 >50 >50 BR07.DG 5.15 4.21 12.85.5 >50 0.898 0.211 >50 >50 HT593.1 1.77 3.86 0.741 0.790 1.510 1.734.51 2.09 2.53 R2 0.931 1.21 0.126 1.490 >50 9.30 0.063 >50 >50BG1168.01 1.52 1.97 >50 2.01 >50 >50 >50 >50 >50 QH0515.01 2.94 2.480.668 1.650 >50 7.23 >50 >50 >50 5768 0.829 0.854 0.995 0.280 >5014.5 >50 1.28 0.580 3988 1.220 0.881 12 1.12 >50 4.14 >50 0.062 0.022Tier 2 Du123.6 >50 >50 >50 >50 >50 9.16 0.938 0.247 0.168 clade CDu151.2 46.5 >50 >50 0.370 2.0 >50 6 0.054 0.016 (n = 34) Du156.12 0.1930.204 >50 0.095 22.8 2.76 >50 0.109 0.019 Du172.17 >50 >50 >50 1.54 >502.62 1.77 0.952 0.147 Du422.1 >50 >50 >50 >50 >50 1.83 >50 1.95 0.924ZM197M.PB7 1.61 2.04 9.23 7.03 >50 >50 >50 2.45 >50 ZM214M.PL15 2.583.19 >50 2.08 >50 40.4 >50 >50 >50 ZM233M.PB6 9.33 4.65 >50 >50 >50 >5012.4 0.016 0.004 ZM249M.PL1 0.232 0.297 >50 0.146 40.1 20.3 >50 0.1490.031 ZM53M.PB12 4.02 4.9 45.6 7.37 0.496 >50 32.2 0.33 0.031 ZM109F.PB40.754 0.619 >50 0.174 0.602 >50 0.281 3.73 >50 ZM135M.PL10a 2.711.59 >50 >50 >50 >50 9.76 >50 >50 CAP45.2.00.G3 >50 >50 >50 >50 >504.09 >50 0.014 0.007 CAP210.2.00.E8 >50 >50 >50 >50 >50 >50 8.3 0.4380.159 CAP244.2.00.D3 2.65 2.08 >50 0.912 >50 >50 17.5 0.341 0.048ZA012.29 1.02 0.654 >50 0.433 >50 >50 45.7 >50 >50 BR025.9 0.5551.1 >50 >50 >50 >50 3.59 0.089 0.019 TV1.29 >50 >50 >50 >50 >50 >50 1.040.036 0.147 ZM215.8 0.527 0.724 >50 0.298 >50 >50 >50 0.437 >50 ZM106.91.02 0.654 >50 0.564 >50 >50 >50 3.56 >50 ZM55.28a 1.2 1.03 >501.92 >50 >50 >50 >50 >50 ZM53.21 3.59 4.01 19.9 4.28 0.327 >50 7.430.071 0.009 ZM55.4a 1.5 1.6 >50 1.53 >50 >50 17.2 41.2 14.5 ZM106.101.37 0.883 0.452 0.421 >50 >50 >50 1.29 2.55 ZM109.32 0.422 0.324 >500.190 0.528 >50 >50 0.727 >50 ZM135.8a 2.052.43 >50 >50 >50 >50 >50 >50 >50 ZM146.7 1.28 1.39 4.48 3.27 >50 >50 >501.69 15.4 ZM176.66 0.258 0.154 0.15 16 >50 >50 35.4 0.036 0.006 ZM181.66.49 3.78 >50 >50 >50 >50 31.6 0.013 0.054 SO18.18 0.178 0.19 0.3240.173 >50 >50 >50 0.106 0.057 286.4 0.839 0.868 18.4 0.836 >50 2.69 39.10.390 0.043 288.4 4 2.56 1.21 1.2 >50 >50 2.05 >50 22.5TZA125.17 >50 >50 >50 >50 >50 >50 39.5 0.666 0.269 TZBD.02 0.328 0.22522.1 0.246 48.6 >50 5.74 1.08 0.101 Clade D UG024.2 0.674 0.619 >500.783 >50 >50 0.025 >50 >50 57128 >50 >50 >50 >50 >50 1.72 0.855 0.63438.3 clade E TH966.8 1.43 1.16 >50 0.437 >50 >50 3.17 0.089 0.015TH976.17 0.486 0.593 >50 0.148 >50 >50 40.8 >50 >50 M02138 1.57 1.99 >501.21 0.157 >50 2.78 1.02 0.553 Non-HIVSIVmac251.30 >50 >50 >50 >50 >50 >50 3.95 >50 >50MuLV >50 >50 >50 >50 >50 >50 >50 >50 >50 Breadth total n = 89 79/8978/89 39/89 71/89 22/89 35/89 56/89 64/89 58/89 (89%) (88%) (44%) (80%)(25%) (39%) (63%) (72%) (65%) clade A n = 24 24/24 24/24  8/24 22/24 6/24  9/24 15/24 23/24 20/24 (100%)  (100%)  (33%) (92%) (25%) (38%)(63%) (96%) (83%) clade B n = 26 24/26 24/26 20/26 22/26  7/26 17/2616/26 10/26 11/26 (92%) (92%) (77%) (85%) (27%) (65%) (62%) (38%) (42%)clade C n = 34 27/34 26/34 11/34 23/34  8/34  8/34 20/34 28/34 24/34(79%) (76%) (32%) (68%) (24%) (24%) (59%) (82%) (71%) Potency* IC50median 0.832 0.665 0.868 0.433 ##### 3.720 6.715 0.192 0.056 IC50geometric mean 0.882 0.739 1.118 0.576 2.934 3.575 5.177 0.256 0.112Red: IC50 or IC80 <1 μg/ml Yellow: IC50 or IC80 between 1 and 1.0 μg/mlGreen: IC50 or IC80 >10 μg/ml

Example 4 Potent and Broad Neutralization by a CD4 Binding SiteMonoclonal Antibody from an HIV-1 Donor Infected with a Clade A1/DRecombinant Virus

Several neutralizing monoclonal antibodies (NAbs) of unprecedentedbreadth and potency, including PG9/16 and VRC01, have been isolated fromHIV-1 positive donors. In this Example, Applicants characterize PGV04(also known as VRC-PG04), a new NAb that has potency and breadth thatrivals that of PG9/16 and VRC01. PGV04 was isolated by single, memory Bcell sorting using a resurfaced core (RSC3) gp120 as bait. The antibodycompeted with CD4, b12 and VRC01 for binding to gp120, confirming ittargets the CD4 binding site (CD4bs). When screened on a large panel ofviruses, PGV04 was distinguished in its neutralizing profile from CD4,b12 and VRC01. In contrast to VRC01, PGV04 did not enhance 17b or X5binding to their epitopes in the co-receptor region on the gp120monomer, and none of the CD4bs monoclonal antibodies (mAbs) were able toinduce the co-receptor site on the functional trimer. When the abilityof PGV04 to neutralize pseudovirus containing single alaninesubstitutions was determined, differences in residue dependence betweenPGV04 and other CD4bs mAbs were revealed. In particular, D279A, I420Aand I423A were found to abrogate PGV04 neutralization. The residuesfound to be important in PGV04 neutralization had varying effects on theability of the antibody to bind gp120 monomer containing the samesubstitutions. Applicants conclude broad and potent CD4bs NAbs havesubtle differences in the way they recognize and access the CD4bs on theviral spike.

A study (Protocol G) that screened 1,800 HIV-1 donors infected withviruses of different clades revealed that a significant fraction ofdonors developed broad and potent neutralizing serum responses inagreement with studies from several laboratories (Doria-Rose et al.;Gnanakaran et al.; Doria-Rose et al. 2009; Gray et al. 2009; Sather etal. 2009; Simek et al. 2009). The top 1% of Protocol G donors thatexhibited the most broad and potent serum neutralizing responses weredesignated “elite neutralizers”. Applicants have previously mapped theserum specificities in 19 Protocol G donors who ranked within the top 5%of donors screened and found the broad serum neutralization of mostdonors was associated with single or a small number of specificities(Walker et al. 2010). The isolation and characterization of broad andpotent neutralizing antibodies from Protocol G donors is a high priorityas the epitopes targeted by these antibodies will facilitate immunogendesign.

There are currently four known regions on the viral spike that aretargeted by potent, broadly neutralizing antibodies (bNAbs). The firstis a conserved region on the primary entry receptor of the virus, theCD4bs. This region is recognized by the bNAbs b12, HJ16, and therecently isolated bNAbs, VRC01 and VRC03 (Corti et al.; Burton et al.1991; Wu et al. 2010). Human bNAbs 2F5, 4E10 and Z13e1 recognize themembrane proximal external region (MPER) on the gp41 protein, anotherconserved region located at the stalk of the viral spike. This region isthought to be important in viral fusion (Ofek et al. 2004; Cardoso etal. 2005; Nelson et al. 2007). The third epitope is composed of acluster of high mannose glycans on the spike, recognized by 2G12, theonly known HIV-1 bNAb to bind solely to glycans (Trkola et al. 1995;Sanders et al. 2002; Scanlan et al. 2002; Calarese et al. 2003). Lastly,PG9 and PG16 recognize a conserved regions of the V1/V2 and V3 loops ongp120 (Walker et al. 2009).

The CD4bs is of particular interest as a conserved region whoseaccessibility, at least to CD4, must be maintained. The first potentbNAb to this region, b12, was isolated from a phage display libraryutilizing RNA from bone marrow lymphocytes of an HIV-1 seropositiveindividual (presumed clade B) (Barbas et al. 1992). The bNAb neutralizes35% of a panel of 162 cross-clade viruses with a median IC₅₀ ofapproximately 3 mg/ml in a pseudovirus assay (Walker et al. 2009).However, b12 solely interacts with gp120 through its heavy chain (Zhouet al. 2007), and the inability to isolate further anti-CD4bs bNAbs ledto doubts as to whether such Abs could be elicited through immunization.A breakthrough was achieved when a bNAb, HJ16, was isolated byimmortalization of memory B cells from a clade C infected donor andshown to exhibit breadth similar to that of b12 (Corti et al.). Morerecently and most significantly, the mAbs VRC01 and VRC03 were isolatedfrom a clade B-infected donor (Wu et al. 2010). VRC01 neutralized 91% ofa panel of 190 pseudoviruses, making it, along with PG9 and PG16, themost broad and potent HIV-1 mAbs isolated to date.

In the present Example, Applicants characterized PGV04, a novel humanCD4bs mAb originating from an elite neutralizer. The antibody wasisolated from single memory B cells in peripheral blood mononuclearcells (PBMC) using the RSC3 protein as bait (Wu et al 2011). The RSC3protein has the antigenic structure of the CD4bs preserved with 30% ofthe surface exposed residues substituted with simian immunodeficiencyvirus (SIV) homologs and other residues differing from the HIV-1sequence. Applicants confirmed that PGV04 is a CD4bs-directed mAb withbroad and potent neutralization capabilities that match those of PG9 andVRC01. Moreover, the neutralizing activity of PGV04 largelyrecapitulated the neutralization profile of the corresponding the donorserum. PGV04 was distinguished from CD4, VRC01 and b12 in its ability toneutralize JR-CSF pseudovirus containing single alanine substitutions.Furthermore, in contrast to VRC01, PGV04 did not enhance binding of theCD4-induced (CD4i) Abs, 17b or X5, to their epitopes colocalized withinthe co-receptor binding site on monomeric gp120, and none of the CD4bsbNAbs induced the CD4i site on functional trimers. Applicants concludefrom these findings that the region of gp120 that composes the CD4bs isable to induce broad and potent mAbs with varying footprints thattranslate into differences in their neutralizing profiles.

Antibodies and Antigens.

The following Abs and reagents were procured by the IAVI NeutralizingAntibody Consortium: 2G12 (Polymun Scientific, Vienna, Austria), X5 and17b (Strategic Biosolutions), soluble CD4, CD4IgG, F425 (provided byLisa Cavacini, Beth Israel Deaconess Medical Center), JR-CSF gp120 andBaL gp120 (provided byGuillaume Stewart-Jones, MRC Human ImmunologyUnit, Oxford), JR-FL gp120 (Progenics, Tarrytown, N.Y.) and YU2 gp120s(provided by Robert Doms, University of Pennsylvania). The RSC3 andΔRSC3 proteins were kindly provided by Richard Wyatt (Scripps, La Jolla,Calif.).

Donor.

The donor from whom PGV04 was isolated was selected from the IAVIsponsored study, Protocol G (Simek et al. 2009). Protocol G enrollmentwas defined as male or female at least 18 years of age with documentedHIV infection for at least three years, clinically asymptomatic at thetime of enrollment, and not currently receiving antiretroviral therapy.High-throughput analytical screening algorithms were used to selectindividuals for mAb generation, and this volunteer was identified as anelite neutralizer based on broad and potent serum neutralizing activityagainst a cross-clade pseudovirus panel.

Binding ELISAS.

RSC3 and ΔRSC3 proteins were diluted in PBS and coated at 2.0 mg/ml andJR-FL gp120 was diluted in PBS and coated at 5.0 mg/ml, 50 ml/well onCostar (3690) 96-well polystyrene ELISA plates overnight at 4° C. Theplates were washed 4× with PBS containing 0.05% tween, and blocked with3% BSA in PBS for 1 hr at 37° C. Then, 5-fold serial dilutions of themAbs, in 1% BSA in PBS, were added at a starting concentration of 10.0mg/ml. The plates were incubated for 1 hr at 37° C. and then washed 4×before the secondary mAb, goat anti-human IgG Fc conjugated to alkalinephosphatase (Jackson) was added at 1:1000 dilution for 1 hr at 37° C.The wells were washed and the signal was detected by adding a 5.0 mgalkaline phosphatase substrate tablet (Sigma) in 5 ml alkalinephosphatase staining buffer (pH 9.8) according to the manufacturer'sinstructions. The optical density at 405 nm was read on a microplatereader (Molecular Devices).

For PGV04 binding to gp120 isolated from JR-CSF pseudovirus, pseudoviruswas collected 3 days post-transfection, supernatants were spun down at300×g for 5 minutes and virus was lysed with 1.0% NP-40 at RT for 30minutes. ELISA plates were coated overnight at 4° C. with an anti-gp120Ab D7324 (International Enzymes, Inc.) at a concentration of 5.0 mg/mlin PBS. Plates were washed 4× and lysed virus was added at 50 ml/welland incubated for 2 hrs at 37° C. Plates were washed 4× and blocked with3% BSA in PBS for 1 hr at RT. PGV04 and 2G12 were added in 5-fold serialdilutions starting at 10.0 mg/ml. Plates were washed 4×, and goatanti-human IgG F(ab′)₂ conjugated to alkaline phosphatase (Pierce) wasadded at a 1:1000 dilution. The remainder of the experiment wasconducted as above.

For competition ELISAs, plates were coated with 5.0 mg/ml of JR-FL gp120in PBS, 50 ml/well overnight at 4° C. Plates were washed 4×, blockedwith 100 ml/well of 3% BSA for 1 hr at RT. Then, 5-fold serial dilutionsof competitor Abs (50 ml/well) were added starting at 10.0-80.0 mg/nildepending on the mAb. The plates were incubated for 30 minutes at RT and50 ml of biotinylated competitor mAb were next added to the solution inthe well at a 50% effective final concentration (EC₅₀). The EC₅₀ isdefined as the concentration at which 50% of the mAb is bound to theprotein. The plate was incubated for 1 hr at RT and washed 4×.Streptavidin-AP was added, 50 ml/well, at a 1:1000 dilution for 1 hr atRT. The plates were washed 4× and the signal was detected using alkalinephosphatase substrate tablets diluted in phosphatase staining buffer asabove.

Induction of the Co-Receptor Binding Site on gp120.

ELISA plates were coated, 50 ml/well, overnight at 4° C. with 5.0 mg/mlof JR-FL or YU2 gp120 protein diluted in PBS. The plates were washed 4×,and blocked with 3% BSA for 1 hr at RT. After removing blockingsolution, 10.0 mg/ml of PGV04, CD4IgG, b12, 2G12, VRC01 or VRC03 wereadded for 30 minutes at RT. Then 5-fold serial dilutions of biotinylatedX5 or 17b (50 ml/well) were added, starting at 50.0 mg/ml and 100.0mg/ml respectively. The plate was washed 4× and 50 ml/well ofstreptavidin conjugated to AP was added at 1:1000 for 1 hr. The platewas washed and developed as above.

Flow Cytometry.

Saturating amounts of PGV04, b12, 2G12, sCD4, VRC01, VRC03 or 17b wereadded at 20.0 mg/ml, 50 ml/well to JR-FL transfected 293T cells seededin 96-well V-bottom plates (Cellstar), and incubated for 30 minutes at4° C. on a plate rotator. Then a 5-fold serial dilution of biotinylated17b starting at 20.0 mg/ml was added, 50 ml/well, to each wellcontaining the competitor mAb for 1 hr at 4° C. on a plate rotator. Theplate was washed 2× and stained with 1:200 dilution of NeutrAvidinconjugated to R-phycoerythrin (PE) (Invitrogen). Binding was analyzedusing flow cytometry, and binding curves were generated by plotting themean fluorescence intensity of antigen binding as a function of antibodyconcentration. A FACSCalibur (BD Biosciences) was used for flowcytometric analysis and FlowJo software for data interpretation.

Neutralization Assays.

Neutralization assays by mAbs and patient sera were performed byMonogram Biosciences as previously described using a single round ofreplication pseudovirus and measuring entry into U87 cells expressingeither CCR5 or CXCR4 by luciferase activity (Richman et al. 2003).Briefly, pseudoviruses were produced by co-transfection of HEK293 cellswith a subgenomic plasmid, pHIV-1lucΔu3, that incorporates a fireflyluciferase indicator gene and a second plasmid, pCXAS that expressesHIV-1 Env libraries or clones. Following transfection, pseudovirus washarvested 3-days later and used to infect U87 cells. The cells werelysed 48 hours post-infection and luciferase activity was read on aluminometer. Generation of pseudoviruses incorporating HIV-1 JR-CSFsingle-alanine substitutions is fully described elsewhere (Pantophlet etal. 2003). Neutralization activity of PGV04 against HIV-1 JR-CSFpseudovirus containing single alanine substitutions in the Env proteinwas measured using a TZM-bl assay as described (Li et al. 2005).

Statistics.

Statistical analyses were done with Prism 5.0 for Mac (GraphPad, LaJolla, Ca).

Characterization of PGV04 as a CD4bs mAb.

PGV04 was isolated from the PBMCs of a clade A infected African donor(Wu et al 2011). Briefly, antigen-specific memory B cells were sortedemploying the RSC3 gp120 to specifically screen for mAbs that target theCD4bs. The ΔRSC3 gp120, in which an amino acid at position 371 wasremoved in order to abolish b12 binding, was used as a negative controlin the sort. As expected, PGV04 binding to the RSC3 gp120 was strong andequivalent to that of VRC01, with both mAbs showing dramaticallydecreased binding to ΔRSC3, consistent with their identification asCD4bs mAbs (FIG. 9A). Similar to VRC01, PGV04 also bound with highaffinity to JRFL gp120 (FIG. 9B).

Applicants next performed competition ELISAs to verify that PGV04 wasdirected against the CD4bs. Serial dilutions of mAbs b12, CD4-IgG,VRC01, 17b, X5, 2G12, or F425 were pre-bound to ELISA plates coated withJRFL gp120 followed by the addition of a constant EC₅₀ concentration ofbiotinylated competitor mAbs listed in the legend of each graph (FIGS.10A-D). PGV04 competed with b12, CD4-IgG and VRC01 for binding to JRFLgp120 confirming that PGV04 targets the CD4bs (FIG. 10A). PGV04 and b12exhibited some partial competition with the CD4i mAbs, 17b and X5 (FIG.10B). However, PGV04 did not compete with 2G12, or the V3-loop directedmAb, F425/b4e8 (FIG. 10C). Competition experiments done in reverse, inwhich serial dilutions of PGV04 were bound to gp120 coated plates andthe EC₅₀ concentrations of the competitor mAbs were subsequently added,gave similar results as expected for reversible binding interactions(FIG. 10D).

Breadth and Potency of Neutralization by PGV04.

To determine the breath and potency of neutralization by PGV04, amulti-clade panel of 162 pseudoviruses was tested in a neutralizationassay using U87 cells expressing either CCR5 or CXCR4 as the target cellline (FIGS. 11A-B). PGV04 neutralized 142 viruses out of the 162-viruspanel with an IC₅₀<50 mg/ml and PG9 neutralized 122 viruses (FIG. 15A;FIG. 11A). The median IC₅₀ of viruses neutralized at an IC₅₀<50 mg/mlwas comparable for PGV04 (0.20 mg/ml) and PG9 (0.27 mg/ml), indicatingPGV04 and PG9 have similar potency. PGV04 neutralized 88% of the virusesin the panel while PG9 neuralized 75% of the viruses in the panel, andthis difference is statistically significant (Fisher's exact test, Pvalue=0.0063) indicating PGV04 is a broader mAb than PG9 (FIG. 11B). Ofnote, there were 7 viral isolates that both mAbs were unable toneutralize but these isolates are known to be neutralized by one of theother existing NAbs: PG16, b12, 4E10, 2G12 and 2F5 (Walker et al. 2009).

Next, Applicants compared the breath and potency of PGV04, PG9 and VRC01on a second 97-pseudovirus panel that used TZM-bl cells as the targetcell line (FIGS. 11A-B and FIG. 16). The results for the two viruspanels were similar, although in the latter panel, the median IC₅₀ ofthe viruses that were neutralized at <50 mg/ml was lower for PG9 thanfor VRC01 and PGV04, and this difference was statistically significant(Mann-Whitney test, P-value <0.0001 and P-value=0.0137 respectively)indicating PG9 (0.06 mg/ml) is more potent on this panel than VRC01(0.17 mg/ml) and PGV04 (0.12 mg/ml). PG9 neutralized 82% of viruses inthe panel, PGV04 neutralized 87% and VRC01 neutralized 93% of viruses inthe 97-virus panel. The difference between PG9 and VRC01 wasstatistically significant (Fisher's exact test, P value=0.0479),indicating VRC01 is a broader antibody than PG9.

The median IC₅₀ for the viruses neutralized with an IC₅₀<50 mg/ml wasslightly lower for PGV04 (0.12 mg/ml) than VRC01 (0.17 mg/ml,Mann-Whitney test, P-value=0.0324) revealing that PGV04 is marginallymore potent than VRC01 on this panel. PGV04 was slightly less broad onthis panel than VRC01 (87% of 97 viruses versus 93%) but this differencewas not statistically significant.

Applicants next investigated the breadth and potency of PGV04 comparedto the donor serum from which PGV04 was isolated. The IC₅₀ of PGV04correlated highly with the 50% neutralization titers (NT₅₀) of the donorserum (Mann-Whitney test, P-value <0.0001). However, there were certaininstances where PGV04 did not neutralize the virus that the donor serumwas able to neutralize (5 out of 162 viruses or 3% of the viruses) (FIG.15). In these cases, Abs distinct from PGV04 appear to be responsiblefor serum neutralizing activity. Surprisingly, in a few cases, PGV04neutralized a particular isolate with an IC₅₀<1 mg/ml while the donorserum did not neutralize this same isolate (7 out of 162 or 4% of theviruses). The reasons for this latter observation are unclear.

Induction of the Co-Receptor Binding Site on Monomeric gp120.

It has been previously shown that soluble CD4 (sCD4) enhances thebinding affinity of the mAb, 17b, to gp120 (Zhang et al. 1999; Zhang etal. 2001). Interestingly, unlike b12, VRC01 has also been shown toenhance 17b binding, although not to the same extent as CD4-IgG (Wu etal. 2010). Applicants were interested in determining whether PGV04binding to gp120 would similarly enhance the binding of CD4i mAbs. Asshown in FIG. 11B, Applicants did not detect enhancement of CD4i mAbbinding in the presence of PGV04. As an alternative to this experiment,Applicants added saturating amounts of PGV04, CD4-IgG, b12, 2G12, VRC01,VRC03 or no mAb to wells coated with monomeric gp120 and then addedtitrating amounts of either biotinylated 17b or X5 (FIGS. 12A-B). Again,PGV04 did not enhance 17b or X5 binding to either JRFL or YU2 gp120,while, CD4-IgG and VRC01 enhanced the binding of both CD4i mAbs.Interestingly, consistant with previously published data, b12 decreased17b and X5 binding suggesting it partially competes with the CD4i mAbs(Moore and Sodroski 1996).

Induction of the Co-Receptor Binding Site on Functional Trimers.

Applicants next investigated whether PGV04 could enable 17b to bind tothe functional trimer. 293T cells were co-transfected with JRFL Env DNA,and pSG3, a plasmid containing the HIV-1 backbone. Forty-eight hourspost-transfection, b12, 2G12, PGV04, sCD4, VRC01, VRC03 or 17b mAbs werepre-bound at saturating concentrations to the surface-expressed Envtrimers. Biotinylated 17b was then titrated onto the pre-bound complex,and binding was detected using flow cytometry. When sCD4 bound gp120 inthe context of the cell surface expressed Env, it created a structuralenvironment that allowed for 17b binding (FIG. 12C). However, no othermAb enabled detectable levels of 17b to bind to the functional trimer.As a control, serial dilutions of biotinylated 2G12 produced identicalbinding curves with each of the pre-bound mAbs, demonstrating that noneof the CD4bs mAbs induced a significant degree of gp120 shedding fromthe cell-surface trimers (FIG. 12D). These results suggest there arestructural constraints in the functional trimer that prevent exposure ofthe co-receptor site on gp120 upon binding of CD4bs mAbs that do notexist in the gp120 monomer.

PGV04 Neutralization of JR-CSF Pseudoviruses Containing Single AlanineSubstitutions.

To map the PGV04 epitope, Applicants performed neutralization assayswith JR-CSF pseudovirus containing single alanine substitutions in thegp120 protein. Substitutions at D279, 1420 and 1423 greatly compromisedPGV04 neutralization, decreasing the neutralization potency to 1%, 3%and 5% that of wildtype respectively (FIG. 13A). D279A also greatlycompromised CD4 and VRC01 neutralization potency yet had little effecton b12 activity. I420A and I423A likewise decreased VRC01 neutralizationpotency although not to the same extent as for PGV04. Moreover, thesetwo substitutions had the opposite effect on b12 and CD4-IgG, increasingthe neutralization potency of both mAbs. Interestingly, the isoleucinesfound at positions 420 and 423 are highly conserved among existing HIV-1viruses in the Los Alamos database, being found at a frequency of 0.97and 0.88 respectively. The aspartic acid found at position 279 is lessconserved and found at a frequency of 0.45, with asparagine being foundat this position at a frequency of 0.51.

Other alanine substitutions also abrogated PGV04 neutralization but notto the same extent as the three residues mentioned above. N276Adecreased PGV04 neutralization potency to 13% that of wild-type.Notably, the N-acetyl-glucosamine from the N-linked glycan at thisresidue has previously been determined to be part of the VRC01 epitopethrough resolution of the crystal structure of VRC01 bound to gp120(Zhou et al.). Surprisingly, removal of this glycan resulted in to a4-fold increase in VRC01 neutralization potency while having no impacton CD4-IgG or b12 neutralization. Additionally, I307A, I309A, F317A, andY318A decreased PGV04 neutralization to 9-36% of wildtype. Theseresidues are found in the tip of the V3 loop and are most likelyimportant in maintaining local structure. Interestingly, these residueshave been shown to be important in maintaining the interaction betweengp120 and gp41 (Xiang et al.), and alanine substitutions at thesepositions have been shown to result in global sensitivity to serumneutralization (Walker et al. 2010). In contrast to PGV04 and (VRC01),these substitutions increased b12 and CD4 neutralization.

PGV04 Binding to JR-CSF gp120 Containing Single Alanine Substitutions.

To further map the PGV04 epitope, Applicants evaluated the bindingactivity of PGV04 to a panel of gp120 proteins containing single alaninesubstitutions that were captured from lysed JR-CSF pseudovirus. D279A(0%), N280A (1%), D457A (4%), and R469A (3%) gp120 variants showedseverely decreased PGV04 binding relative to wild-type gp120, suggestingthat the residues involved are part of the PGV04 epitope (FIG. 13B). Theresult for D279A gp120 is consistent with the neutralization data foundfor the D279A variant virus in FIG. 13A. Further, three potentialresidues are added to the PGV04 epitope map that could not be tested inthe neutralization experiments because the corresponding variant virusinfectivity values were below the detection level of the assay. One ofthe substitutions, R469A, is found in the V5 loop and also decreases2G12 binding. Therefore this substitution most likely disrupts gp120structure and is not directly part of the PGV04 epitope. The D457Asubstitution has previously been shown to decrease binding to below 10%of wildtype for b12 and CD4 (Pantophlet et al. 2003) and D368A whichdecreases PGV04 binding to 12%, has previously been shown to be part ofthe CD4, b12 and VRC01 epitopes but not that of HJ16 (Corti et al.; Zhouet al.; Walker et al. 2010).

PGV04 Binding to Deglycosylated BaL gp120.

In Applicants' alanine scanning studies, Applicants found certainN-linked glycans affected PGV04 binding and neutralization. Therefore,Applicants determined whether the removal of glycans by Endo H, Endo F1,Endo F2, and Endo F3 from BaL gp120 protein, produced in 293T cells,would affect binding of PGV04. PGV04 and the other CD4bs mAbs, b12,VRC01, VRC03 and b6, retained similar levels of binding to both mock anddeglycosylated forms of the protein suggesting CD4bs mAbs in general donot have a strong dependence on glycans for binding to gp120 (FIGS.14A-B).

In this Example, Applicants characterized PGV04, a new broad and potentCD4bs-directed HIV-1 neutralizing mAb. PGV04 matched the breath andpotency of PG9 and VRC01, two previously described potent HIV-1 bNAbs.PG9 and PGV04 neutralization was determined on two different pseudoviruspanels. For a 162-pseudovirus panel in U87 cells, PGV04 and PG9 hadcomparable potency, with PGV04 having greater breadth. For a different97-pseudovirus panel in TZM-bl cells, a slightly different outcomeresulted. PG9 was more potent than PGV04, and PG9 had similar breadth asPGV04. PG9 neutralized the viruses in the latter panel with greaterpotency than the viruses in the 162-virus panel, while, PGV04 seemed tohave similar neutralization potency on both panels. This may be due tothe isolates chosen on both panels and/or the target cell line used, U87versus TZM-bl cells. PGV04 had higher breadth than PG9 on both panelsalthough this difference was statistically significant only on the162-virus panel.

Overall, PG9/16, VRC01 and PGV04 neutralized more than 70% ofcirculating viruses with 10-fold greater potency than the earlierestablished HIV-1 bNAbs, b12, 2G12, 2F5, and 4E10. Interestingly, thevirus of the PGV04 donor was subtyped as clade Al/D recombinant, whichdiffers from the donors associated with the other CD4bs bNAbs, VRC01(clade B), HJ16 (clade C) and b12 (presumed clade B). Therefore, itseems that elicitation of broadly neutralizing CD4bs-directed mAbs isnot dependent on the clade of the infecting isolate. Of note, the PGV04donor virus does not appear to be of any known circulating recombinantform (CRF) listed in the Los Alamos HIV databases.

Applicants' results show significant differences between CD4bs bNAbs intheir mode of recognition of the CD4bs region. For example, CD4-IgG andVRC01 enhance exposure of the co-receptor binding site on monomericgp120, while PGV04 and b12 do not. Moreover, VRC01, b12 and PGV04, incontrast to CD4, did not induce the co-receptor site on functionaltrimers. These observations suggest that differences exist between thepresentation of the CD4bs in recombinant gp120 versus gp120 in thecontext of the functional trimer. These observations also highlightdifferences in VRC01 and PGV04 recognition of the CD4bs, as it isexposed on monomeric gp120.

In Applicants' alanine scanning studies, several alanine substitutionsaffected the CD4bs mAbs differently, again illustrating that PGV04,VRC01, b12 and CD4-IgG recognize the CD4bs in somewhat different ways.For example, D279A, I420A and I423A substitutions greatly decreasedneutralization of PGV04, but varied in their effects on VRC01, CD4-IgGand b12. The D279A substitution decreased neutralization by VRC01 andCD4-IgG but had no great effect on b12. The I420A and I423Asubstitutions decreased VRC01 neutralization but increased both CD4-IgGand b12 neutralization. In addition, certain substitutions in the V3loop decreased neutralization by PGV04 and VRC01 but increasedneutralization by CD4 and b12. Importantly, the highly conserved natureof the residues important for PGV04 recognition likely explains howPGV04 is able to achieve broad neutralization. Indeed, it will beinteresting to determine which residues are important for natural escapeto PGV04.

The results of this Example have implications for vaccine design.Considering that a collection of broad and potent CD4bs-directedneutralizing antibodies have been isolated from naturally infecteddonors, and CD4bs neutralizing activity has been detected in severalbroadly neutralizing sera (Li et al. 2007; Binley et al. 2008; Gray etal. 2009; Li et al. 2009; Sather et al. 2009), it appears that thisregion may be a particularly promising vaccine target. Also Applicants'data show that different CD4bs bNAbs can target slightly differentepitopes, which may have implications for the design of immunogens thatfocus the immune response on the CD4bs.

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Example 5 Focused Evolution of HIV-1 Neutralizing Antibodies Revealed byCrystal Structures and Deep Sequencing

Antibody VRC01 is a human immunoglobulin that neutralizes ˜90% of HIV-1isolates. To understand how such broadly neutralizing antibodiesdevelop, Applicants used X-ray crystallography and 454 pyrosequencing tocharacterize additional VRC01-like antibodies from HIV-1-infectedindividuals. Crystal structures revealed a convergent mode of bindingfor diverse antibodies to the same CD4-binding-site epitope. Afunctional genomics analysis of expressed heavy and light chainsrevealed common pathways of antibody-heavy chain maturation, confined tothe IGHV1-2*02 lineage, involving dozens of somatic changes, and capableof pairing with different light chains. Broadly neutralizing HIV-1immunity associated with VRC01-like antibodies thus involves theevolution of antibodies to a highly affinity-matured state required torecognize an invariant viral structure, with lineages defined fromthousands of sequences providing genetic roadmaps of their development.

HIV-1 exhibits extraordinary genetic diversity and has evolved multiplemechanisms of resistance to evade the humoral immune response (1-3).Despite these obstacles, 10-25% of HIV-1-infected individuals developcross-reactive neutralizing antibodies after several years of infection(4-9). Elicitation of such antibodies could form the basis for aneffective HIV-1 vaccine, and intense effort has focused on identifyingresponsible antibodies and delineating their characteristics. A varietyof monoclonal antibodies (mAbs) have been isolated that recognize arange of epitopes on the functional HIV-1 viral spike, which is composedof three highly glycosylated gp120 exterior envelope glycoproteins andthree transmembrane gp41 molecules. Some broadly neutralizing antibodiesare directed against the membrane-proximal external region of gp41(10-11), but the majority recognize gp120. These include the quaternarystructure-preferring antibodies PG9, PG16, and CH01-04 (12-13), theglycan-reactive antibodies 2G12 and PGT121-144 (14-15), and antibodiesb12, HJ16, and VRC01-03, which are directed against the region of HIV-1gp120 involved in initial contact with the CD4 receptor (16-19).

One unusual characteristic of all these gp120-reactive broadlyneutralizing antibodies is a high level of somatic mutation. Antibodiestypically accumulate 5-15% changes in variable domain-amino acidsequence during the affinity maturation process (20), but for thesegp120 reactive neutralizing antibodies, the degree of heavychain-somatic mutation is markedly increased, ranging from 19% for thequaternary structure-preferring antibodies (12), to 31% for antibody2G12 (21-22), and to 40-46% for the CD4-binding-site antibodies, HJ16(17), VRC01, VRC02, and VRC03 (18).

In the case of VRC01, the mature antibody accumulates roughly 70 totalchanges in amino acid sequence during the maturation process. The matureVRC01 can neutralize ˜90% of HIV-1 isolates at a geometric mean IC₅₀ of0.3 μg/ml (18), and structural studies show that it achieves thisneutralization by precisely recognizing the initial site of CD4attachment on HIV-1 gp120 (19). By contrast, the predicted unmutatedgermline ancestor of VRC01 has weak affinity for typical strains ofgp120 (˜mM) (19). Moreover, with only three VRC01-like antibodiesidentified in a single individual (donor 45), it has been unclearwhether the VRC01-mode of recognition, genetic origin, and pathway ofaffinity maturation represent general features of the B-cell response tothe CD4-binding site of HIV-1 gp120. Here Applicants explore how broadlyneutralizing HIV-1 immunity associated with VRC01-like antibodiesdevelops, with an analysis of dozens of neutralizers from additionaldonors to answer questions of generality and to trace pathways ofaffinity maturation with thousands of VRC01-like antibody sequences.

Isolation of Neutralizing Antibodies from Donors 74 and 0219 with aCD4-Binding-Site Probe.

Applicants previously used structure-guided resurfacing to alter theantigenic surfaces on HIV-1 gp120 while preserving the initial site ofattachment to the CD4 receptor (18). With the resurfaced stabilized core3 probe (RSC3), over 30% of the surface residues of core gp120 werealtered and the conformation stabilized by the addition ofinterdomain-disulfide bonds and cavity-filling point mutations (18).Applicants used RSC3 and a mutant version containing a single amino aciddeletion in the CD4-binding loop (ΔRSC3) to interrogate a panel of 12broadly neutralizing sera derived from the IAVI protocol G cohort ofHIV-1 infected individuals (6, 23) (FIG. 17A). A substantial fraction ofneutralization of three sera was specifically blocked by RSC3 comparedwith A RSC3, indicating the presence of CD4-binding-site-directedneutralizing antibodies. RSC3-neutralization competition assays alsoconfirmed the presence of CD4-binding-site antibodies in the previouslycharacterized sera 0219, identified in the CHAVI 001 cohort (8) (FIG.17A). Peripheral blood mononuclear cells (PBMCs) from protocol G donor74 (infected with AD recombinant) and from CHAVI donor 0219 (infectedwith clade A) were used for antigen-specific B-cell sorting and antibodyisolation. For donor 74 and 0219, respectively, a total of 0.13% and0.15% of IgG⁺ B cells were identified (FIG. 17B). The heavy and lightchain immunoglobulin genes from individual B-cells were amplified andcloned into IgG1 expression vectors that reconstituted the full IgG (18,24). From donor 74, two somatically related antibodies named VRC-PG04and VRC-PG04b demonstrated strong binding to several versions of gp120and to RSC3 but >100-fold less binding to ΔRSC3. From donor 0219, fivesomatically related antibodies named VRC-CH30, 31, 32, 33 and 34displayed a similar pattern of RSC3; ΔRSC3 reactivity. Sequence analysisof these two sets of antibody clones (FIG. 17C) revealed that theyoriginated from the same inferred immunoglobulin heavy chain variable(IGHV) precursor gene allele IGHV1-2*02. Despite this similarity inheavy chain V-gene origin, the two clone sets originated from differentheavy chain J segment genes and contained different light chains. Thelight chains of the VRC-PG04 and 04b somatic variants originated from anIGκV3 allele whereas the VRC-CH30-34 somatic variants derived from anIGκV1 allele. Of note, all seven antibodies were highly affinitymatured: VRC-PG04 and 04b displayed a V_(H) gene mutation frequency of30% relative to the germline IGHV1-2*02 allele, a level of affinitymaturation similar to that previously observed with VRC01-03; theVRC-CH30-34 antibodies were also highly affinity matured, with V_(H)mutation frequency of 23-25%.

To define the reactivities of these new antibodies on gp120, Applicantsperformed competition ELISAs with a panel of well-characterized mAbs.Binding by each of the new antibodies was competed by VRC01-03, by otherCD4-binding-site antibodies and by CD4-Ig, but not by antibodies knownto bind gp120 at other sites. Despite similarities in gp120 reactivityand V_(H)-genomic origin, sequence similarities of heavy and light chaingene regions did not readily account for their mode of gp120recognition. Finally, assessment of VRC-PG04 and VRC-CH31 neutralizationon a panel of Env-pseudoviruses revealed their ability to potentlyneutralize a majority of diverse HIV-1 isolates (FIG. 17D).

Structural Definition of Gp120 Recognition by RSC3-Identified Antibodiesfrom Different Donors: a Remarkable Convergence.

To define the mode of gp120 recognition employed by donor 74-derivedVRC-PG04, Applicants crystallized its antigen-binding fragment (Fab) incomplex with a gp120 core from the clade A/E recombinant 93TH057 thatwas previously crystallized with VRC01 (19). Diffraction data to 2.1 Åresolution were collected from orthorhombic crystals, and the structuresolved by molecular replacement and refined to a crystallographicR-value of 19.0% (FIG. 18A). The structure of VRC-PG04 in complex withHIV-1 gp120 showed striking similarity with the previously determinedcomplex with VRC01, despite different donor origins and only 50% aminoacid identity in the heavy chain-variable region (FIG. 18). When gp120swere superimposed, the resultant heavy chain positions of VRC-PG04 andVRC01 differed by a root-mean-square deviation (rmsd) of 2.1 Å inCα-atoms, with even more precise alignment of the heavy chain-secondcomplementarity determining (CDR H2) region (1.5 Å rmsd). Criticalinteractions such as the Asp368_(gp120) salt bridge to Arg71_(VRC01)were maintained in VRC-PG04 (FIG. 18B).

Applicants also crystallized the gp120-Fab complex of the donor45-derived VRC03 mAb, the isolation and initial characterization ofwhich were previously described (18). VRC03 and VRC-PG04 share only 51%heavy chain-variable protein sequence identity, and the heavy chain ofVRC03 contains an unusual insertion in the framework 3 region (18).Diffraction data to 1.9 Å resolution were collected from orthorhombiccrystals, and the structure solved by molecular replacement and refinedto a crystallographic R-value of 18.7% (FIG. 18). VRC03 also showedrecognition of gp120 that was strikingly similar to that of VRC-PG04 andVRC01, with similar interface residues and pairwise rmsds in Cα-atoms of2.4 and 1.9 Å, respectively. In particular, gp120-interactive surfacesof CDR H2 and CDR L3 showed similar recognition (pairwise Cα-rmds ofthese regions of the antibodies ranged from 0.5-1.4 Å aftersuperposition of gp120).

In general, the repertoire of possible immunoglobulin products is verylarge and highly similar modes of antibody recognition are expected tooccur infrequently (25). To assess how atypical the VRC01-like antibodyconvergence was, Applicants analyzed other families of HIV-1 specificantibodies that share common IGHV-gene origins (26-29), including theCD4-induced antibodies, 17b, 412d and X5, all of which derive from acommon IGHV1-69 allele. Analysis of the recognition of gp120 by theseantibodies indicated substantial variation, with angular difference inheavy chain orientation between 17b, 412d and X5 of over 90°, or roughly10-fold greater than among the VRC01-like antibodies. Also, the RSC3probe may select for a particular mode of recognition, so Applicantsanalyzed other CD4-binding-site antibodies that bind strongly to theRSC3 probe, including antibodies b12 and b13 (16, 30); these otherRSC3-reactive antibodies also showed dramatic differences in heavy chainorientation relative to the VRC01-like antibodies.

The remarkable convergence in recognition observed with VRC01, VRC03,and VRC-PG04 suggested a common mode of HIV-1 gp120 recognition,conserved between donors infected with a clade B (donor 45) or a cladeA/D (donor 74) strain of HIV-1. The precision required for this mode ofrecognition likely arises as a consequence of the multiple mechanisms ofimmune evasion that protect the site of CD4 attachment on HIV-1 gp120(30). Applicants analyzed paratope surface properties and found that theaverage energy of antibody hydrophobic interactions (Δ^(i)G) correlatedwith the convergence in antibody recognition (P=0.0427) (FIG. 19A) (31).Thus although precise H-bonding is required for this mode of recognition(FIG. 18C), the convergence in structure appears to optimize regionswith hydrophobic interactions. Another important feature of this mode ofrecognition is its ability to focus precisely on the initial site of CD4receptor attachment (19, 32). Indeed, the breadth of HIV-1neutralization among CD4-binding-site ligands correlated with targetingonto this site (P=0.0405) (FIG. 19B).

This convergence in epitope recognition is accompanied by a divergencein antibody sequence identity (FIGS. 17C AND 19C). All ten antibodiesisolated by RSC3 binding utilize the IGHV1-2*02 germline and accrue70-90 nucleotide changes. Despite this similarity in the epitoperecognized by these mature antibodies, only two residues from thegermline IGHV1-2*02 allele mature to the same amino acids (FIG. 17C).Both of these changes occur at a hydrophobic contact in the critical CDRH2 region (56: Gly→Ala and 57: Thr→Val). The light chains for donors 45and 74 antibodies arise from either IGVκ3-11*01 or IGVκ3-20*01, whereasthe light chains of donor 0219 antibodies are derived from IGVκ1-33*01.For these light chains, no maturational changes are identical. Despitethis diversity in maturation, comparison of the VRC01, VRC03, andVRC-PG04 paratopes shows that many of these changes are of conservedchemical character (FIG. 19C); a hydrophobic patch in the CDR L3, forexample, is preserved. These observations suggest that divergent aminoacid changes among VRC01-like antibodies nevertheless afford convergentrecognition when guided by affinity maturation.

Functional complementation of heavy and light chains among VRC01-likeantibodies.

Although the identification and sorting of antigen-specific B cells withresurfaced probes has resulted in the isolation of several broadlyneutralizing antibodies, genomic analysis of B-cell cDNA librariesprovides thousands of sequences for analysis. These sequences specifythe functional ‘antibodyome’, the repertoire of expressed antibody heavyand light chain sequences in each individual. High-throughput sequencingmethods provide heavy chain and light chain sequences, but do not retaininformation about their pairings. For VRC01-like antibodies, thestructural convergence revealed by the crystallographic analysisindicated a potential solution: different heavy and light chains mightachieve functional complementation within this antibody family.

Heavy and light chain chimeras of VRC01, VRC03, VRC-PG04 and VRC-CH31were produced by transient transfection and tested for HIV-1neutralization. VRC01 (donor 45) and VRC-PG04 (donor 74) light chainswere functionally compatible with VRC01, VRC03 and VRC-PG04 heavychains, though the VRC03 light chain was compatible only with the VRC03heavy chain (FIG. 20A). Similarly, despite ˜50% differences in sequenceidentity, the VRC-CH31 (donor 0219) heavy and light chains were able tofunctionally complement most of the other antibodies (FIG. 20A).

Identification of VRC01-Like Antibodies by Deep Sequencing of Donors 45and 74.

To study the antibody repertoire in these individuals, Applicantsperformed deep sequencing of cDNA from donor 45 PBMC (33). Because thevariable regions of heavy and light chains are roughly 400 nucleotidesin length, 454 pyrosequencing methods, which allow read lengths of 500nucleotides, were used for deep sequencing. Applicants first assessedheavy chain sequences from a 2008 PBMC sample from donor 45, the sametime point from which antibodies VRC01, VRC02, and VRC03 were isolatedby RSC3-probing of the memory B-cell population (18). mRNA from 5million PBMC was used as the template for PCR to preferentially amplifythe IgG and IgM genes from the IGHV1 family. The 454 pyrosequencingprovided 221,104 sequences of which 33,386 encoded heavy chain variabledomains that encompassed the entire V(D)J region (Appendix 1). Tocategorize the donor 45-heavy chain sequence information, Applicantschose characteristics particular to the heavy chains of VRC01 and VRC03as filters: (i) sequence identity, (ii) IGHV gene allele origin, and(iii) sequence divergence from the germline IGHV-gene as a result ofaffinity maturation (FIG. 20B). Specifically, Applicants dividedsequences into IGHV1-2*02 allelic origin (4,597 sequences) andnon-IGHV1-2*02 origin (28,789 sequences), and analyzed divergence frominferred germline genes, and sequence identity to the templateantibodies VRC01 and VRC03 (FIG. 20B). Interestingly, no sequence ofhigher than 75% identity to the VRC01 or VRC02 heavy chain was found(FIG. 20B), although 109 sequences of greater than 90% sequence identityto VRC03 were found and all were of IGHV1-2*02 origin. These heavy chainsequences formed a well segregated cluster on a contour plot (FIG. 20B,top middle panel). Applicants next assessed the biological function oftwo randomly selected heavy chain sequences from this cluster. Chimericantibodies were made by pairing each of the two heavy chain sequenceswith the VRC03 light chain. In both cases, potent neutralization wasobserved, with neutralization similar to the original VRC03 antibody(FIG. 20E) (34).

A similar heavy chain-deep sequencing analysis was performed with donor74 PBMC from the same 2008 time point from which VRC-PG04 and VRC-PG04bwere isolated. In the initial analysis, despite obtaining 263,764sequences of which 85,851 encompassed the full V(D)J regions of theheavy chain and 93,112 were unique, no sequences of greater than 75%nucleotide identity to VRC-PG04 were found. Because the number of uniqueheavy chain mRNAs present in the PBMC sample was likely much larger thanthe number of unique sequences obtained in the initial analysis,Applicants repeated the deep sequencing of this sample with an increasednumber of 454 pyrosequencing reads and with protocols that optimizedread length. In this analysis, 110,386 sequences of IGHV1-2*02 originand 606,047 sequences of non-IGHV1-2*02-origin were found to encompassthe V(D)J region of the heavy chain, a 10-fold increase in sequencingdepth. Among these sequences, 4,920 displayed greater than 75%nucleotide identity to VRC-PG04 (FIG. 20B). Heavy chain sequences of theIGHV1-2*02 allelic origin segregated into several clusters, one at ˜25%divergence and ˜85% identity to the VRC-PG04 heavy chain, and several at25-35% divergence and 65%, 85%, and 95% identity to VRC-PG04 (FIG. 20B,top right panel). To assess the biological function of these numerous454-identified heavy chain sequences, Applicants selected 56representative sequences from the quadrant defined by high divergence(16-38%) and high sequence similarity (60-100%) to VRC-PG04. The 56sequences were synthesized and expressed with the VRC-PG04 light chain).Remarkably, many of these antibodies displayed potent HIV-1neutralization (35), confirming that these were functional VRC-PG04-likeheavy chains (FIG. 20E).

Applicants next performed a similar analysis of the antibody lightchain. Because VRC01-03 and VRC-PG04 derive from IGκV3 alleles,Applicants used primers designed to amplify the IGκV3 gene family.Applicants chose a donor 45 2001 time point to maximize the likelihoodof obtaining light chain sequences capable of functional complementation(36). A total of 305,475 sequences were determined of which 87,658sequences encompassed the V-J region of the light chain (Appendix 4). Toclassify the donor 45-light chain sequences into useful subsets,Applicants again chose biologically specific characteristics: Adistinctive 2-amino acid deletion in CDR L1 and high affinity maturation(17% and 19% for VRC01 and VRC-PG04, respectively). Two such sequenceswith ˜90% sequence identity to the VRC01 and VRC03 light chainsrespectively, were identified (FIG. 20C). Applicants assessed thebiological function of these two light chains after synthesis andexpression in combination with the VRC01, VRC03, and VRC-PG04 heavychains. When paired with their respective matching wild type heavy chainto produce a full IgG, both chimeric antibodies displayed neutralizationsimilar to the wild type antibody (FIG. 20D).

Maturation Similarities of VRC01-Like Antibodies in Different DonorsRevealed by Phylogenetic Tools.

The structural convergence in gp120 recognition and the functionalcomplementation between VRC01-like antibodies from different donorssuggested similarities in their maturation processes. Applicantstherefore used well-established phylogenetic tools to assess theevolutionary relationship among sequences derived from the sameprecursor germline gene (37). Applicants hypothesized that if knownVRC01-like sequences from one donor were added to the analysis ofsequences of another donor, the resultant ‘cross-donor phylogenetic’analysis might reveal similarities in antibody maturation pathways.Specifically, with such an analysis, the exogenous sequences would beexpected to interpose between dendrogram branches contain VRC01-likeantibodies from the original donor's antibodyome. Applicants performedthis analysis with heavy chains, as all of the probe-identifiedVRC01-like antibodies derived from the same heavy chain IGHV1-2*02allele. Applicants added the donor 74-derived VRC-PG04 and 4b and donor0219-derived VRC-CH30-32 heavy chain sequences to the donor 45 heavychain sequences of IGHV1-2*02 genomic origin and constructed a treerooted by the predicted VRC01-unmutated germline ancestor (18). Thisanalysis revealed that sequences of high identity to VRC03 clustered asa subtree of a common node that was also the parent to donor 74 and 0219VRC01-like heavy chain sequences (FIG. 21A, left). Two donor 45sequences chosen at random from the subtree derived from this commonnode were shown to neutralize HIV-1, whereas 11 heavy chain sequencesfrom outside this node did not neutralize (P<0.0001).

Applicants also assessed the donor 74-derived IGHV1-2*02 heavy chainsequences by including probe-identified VRC01-like antibodies from donor45 and donor 0219 in the cross-donor phylogenetic analysis. In the treerooted by the predicted VRC-PG04 unmutated germline ancestor, 5,047sequences segregated within the donor 45 and 0219-identified subtree(FIG. 21A, right). This subtree included the actual VRC-PG04 and 04bheavy chain sequences, 4,693 sequences of >85% identity to VRC-PG04, andseveral hundred sequences with identities as low as 68% to VRC-PG04. Totest the functional activity of heavy chain sequences identified by thisanalysis, Applicants first assessed the tree location of the 56 heavychain sequences that were identified and expressed from the previouslydescribed identity/divergence grid (FIG. 22A). To these 56 sequences,Applicants added 7 additional sequences from the donor 74 tree and 7non-IGHV1-2*02 sequences to enhance coverage of the cross-donorsegregated sequences (FIG. 22B). These 70 sequences were synthesized andexpressed with the VRC-PG04 light chain (FIG. 22C). Among these 70synthesized heavy chain sequences, 25 did not express. Of the remaining45 reconstituted antibodies, 24 were able to neutralize HIV-1 (FIG.22B). Remarkably, all of the neutralizing sequences segregated into thesubtree identified by the exogenously added donor 45 and 0219 VRC01-likeantibodies (P-value=0.0067) (FIG. 22D).

Applicants also applied this cross-donor segregation method to the lightchains antibodyome of donor 45. The light chains from donors 74 and 0219did not segregate with known VRC01-like light chains from donor 45,likely because these three light chains do not arise from the sameinferred germline sequences. This difference may also reflect thedissimilarities in focused maturation of the two chains (see FIG. 19A):in the heavy chain, focused maturation occurs in the CDR H2 region(encompassed solely within the IGHV1-2*02 V_(H) gene from which allVRC01-like heavy chains derive) and, in the light chain, selectionpressures occur in the CDR L3 region (which is a product of differenttypes of V-J recombination).

CDR H3-Lineage Analysis.

The 37 heavy chain sequences that both segregated into the VRC01neutralizing subtree and expressed when reconstituted with the VRC-PG04light chain could be clustered into 9 CDR H3 classes (FIG. 22B), withsequences in each class containing no more than 5 nucleotide differencesin CDR H3 from other sequences in the same class. A detailed junctionanalysis of the V(D)J recombination origins of these classes suggestedthat 8 of the 9 classes arose by separate recombination events; two ofthe classes (7 and 8) differed primarily by a single three-residueinsertion deletion, Arg-Tyr-Ser, and may have arisen from a single V(D)Jrecombination event. Three of these classes (CDR H3-1, 2, and 9) wererepresented only by non-neutralizing antibodies, three by a singleneutralizing antibody (CDR H3-4, 5 and 6), and three by a mixtures ofneutralizing and non-neutralizing antibodies (CDR H3-3, 7 and 8) (38).Although it was not clear if the non-neutralizing heavy chain sequencestruly lacked neutralization function or if this phenotype was due toincompatibilities in light chain pairing, Applicants chose to analyzeCDR H3 classes only for those in which neutralization had beenconfirmed.

Applicants further analyzed donor 74 IGHV1-2*02 heavy chain sequences toprovide an overview of CDR H3 diversity relative to sequence identityand divergence (FIG. 22E) and to identify those with CDR H3 sequencesidentical to the CDR H3s in each of the neutralizing classes. Thisanalysis identified four clonal lineages (CDR H3-classes 3, 6, 7 and 8),with sequences that extended to 15% or less affinity maturation. CDR H3class 7 included the probe-identified antibodies, VRC-PG04 and 04b (FIG.22B). In each case, a steady accumulation of changes in both frameworkand CDR regions led to increased neutralization activity (39), andchanges at positions 48, 52, 58, 69, 74, 82 and 94 in the V gene, amongothers, appeared to be selected in several lineages. Overall, more than1,500 unique sequences could be classified into these four CDR H3lineages. Although these CDR H3 lineages were inferred from a singletimepoint, they likely provide insight into the specific maturationpathways by which the heavy chains of VRC01-like antibodies evolve froman initial unmutated recombinant to a broadly neutralizing antibody.

J Chain Analysis and Maturation Complexities.

Among the heavy chain VRC01-like sequences identified in donors 45 and74, a significant skewing of J chain usage was observed (FIG. 21A): indonor 45, over 87% of the cross donor-segregated sequences utilize theIGHJ1*01 allele, and in donor 74, 99% of the segregated sequencesutilize the IGHJ2*01 allele. This preferential heavy J chain usage doesnot appear to be a requirement for binding specificity; indeed, the useof the J1 allele in VRC01, the J2 allele in VRC-PG04, and the J4 allelein VRC-CH31 provide examples for the functional compatibility of atleast three different IGHJ alleles in VRC01-like antibodies. In additionto preferential J chain usage, other complexities in the maturationprocess could be inferred from similarities in mature heavy chain genesand differences in CDR H3 sequence. In the absence of information on thenatural pairing of heavy and light chains, the antibody maturationprocesses underlying these complexities is difficult to infer.Nevertheless, the deep sequencing data, with thousands of CDR H3-definedmaturation intermediates, provide sufficient information to suggest thatthe maturation process may involve heavy chain revision or othermechanisms of B cell diversification (40, 41).

Antibody Genomics, HIV-1 Immunity, and Vaccine Implications.

Affinity maturation that focuses a developing antibody onto a conservedsite of HIV-1 vulnerability provides a mechanism to achieve broadrecognition of HIV-1 gp120. Such focused evolution may be common tobroadly neutralizing antibodies that succeed in overcoming the immuneevasion that protects HIV-1 gp120 from humoral recognition; the multiplelayers of evasion may constrain or focus the development of nascentantibodies to particular pathways during maturation.

The structure-based genomics approach described provides tools forunderstanding antibody maturation. Applicants show how deep sequencingcan be utilized to determine the repertoire of specific families ofheavy and light chain sequences in HIV-1 infected individuals. Thesepartial antibodyomes can then be interrogated for unusual properties insequence, or in maturation, to identify antibodies for functionalcharacterization. Applicants demonstrate three means of sieving a largedatabase of antibody sequences: 1) by identity to a known mAb sequenceand by divergence from putative germline (identity/divergence-gridanalysis), 2) by cross-donor phylogenetic analysis of maturation pathwayrelationships, and 3) by CDR H3-lineage analysis. These three means ofsieving can be deployed both iteratively or in combination (FIG. 22). Animportant aspect of Applicants' analyses was the functionalcharacterization of selected sequences achieved through expression ofand reconstitution with known VRC01-like heavy or light chains, althoughother means of pairing such as by frequency analysis (42) are possible.Although neutralization has been assessed on less than 100 reconstitutedantibodies, the thousands of identified heavy and light chain sequencesprovide a large dataset for analysis, which should enhance Applicants'understanding of the critical features of VRC01-like antibodies. Forexample, the correlation of sequence variation at particular positionswith neutralization should provide insight into the allowed diversityand required elements of neutralization by this family of antibodies.

The deep sequencing and structural bioinformatics methodologiespresented here facilitate analysis of the human antibodyome. Thisgenomics technology allows interrogation of the antibody responses frominfected donors, uninfected individuals, or even vaccine recipients andhas several implications. For example, a genomic rooted analysis of theVRC01 antibodyome with standard phylogenetic tools may reveal a generalB cell maturation pathway for the production of VRC01-like antibodies.Indeed, cross-donor phylogenetic analysis (FIG. 21B) suggests thatcommon maturation intermediates with 20-30 affinity maturation changesfrom the IGHV1-2*02 genomic precursor are found in differentindividuals. These intermediates give rise to mature, broadlyneutralizing VRC01-like antibodies, which have about 70-90 changes fromthe IGHV1-2*02 precursor (FIG. 21B). If modified gp120s with affinity tothe maturation intermediates represented by the nodes of the tree wereto stimulate the elicitation of these intermediates, then the analysispresented here can help guide the vaccine-induced elicitation ofVRC01-like antibodies. Deep sequencing not only provides a means toidentify such intermediates, but also a means to facilitate theirdetection. Overall, the application of genomic technologies to analysisof antibodies facilitates both highly sensitive feedback and anunprecedented opportunity to understand the response of the antibodyometo infection and vaccination.

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34. Applicants also assessed 454-derived sequences for structuralcompatibility with the VRC01, VRC03, and VRC-PG04 gp120-complex crystalstructures using a threading algorithm which assessed structuralcompatibility using the DFIRE statistical potential (43). None of theten sequences with optimal DFIRE scores, nor those with high germlinedivergence of non-IGHV1-2*02 genomic origin displayed neutralizationwhen reconstituted with the VRC01 light chain (FIG. 20E). Thus, sequencesimilarity, IGHV1-2*02 origin, and divergence all correlate withneutralization potential, but other factors such as predicted structuralcompatibility failed to identify VRC01-like antibodies.

35. Six of the reconstituted antibodies displayed a mean IC₅₀ of ˜0.1μg/ml, a level of potency similar to that observed with the originalprobe-identified VRC-PG04 antibodies.

36. 1) VRC03L does not complement well with other heavy chains; 2) VRC03H was readily found among donor 45 2008 sequences; 3) VRC01 and VRC02 Hwere not found among donor 45 2008 sequences; 4) VRC01-03 were isolatedfrom the memory B-cell population. Results 1-4 suggests that VRC03 cameafter VRC01; Applicants therefore choose a pre-2008 timepoint tomaximize chances of obtaining light chains that allowed for functionalcomplementation with known VRC01 heavy chains.

37. Although phylogenetic analysis is often used to study the evolutionof a family of sequences and to understand the relationships betweenancestral sequences and their descendants, Applicants appreciate thatthere are some unique aspects to antibody evolution. Due to the natureof activation-induced cytidine deaminase (AID) activity, antibodiesaccumulate mutations at hotspots (CDRs) and thus do not occur is astochastic manner throughout the antibody genome. Also, the process ofVDJ recombination introduces nucleotide insertions and deletions thatalter germline DNA sequence. Applicants' goal here was to elucidate theontogeny of recombined antibody sequences in order to identifyintermediate sequences related to mature neutralizing antibodies.Applicants therefore used well established maximum likelihoodphylogenetic algorithms to analyze antibody sequence data and to buildrooted trees of antibody sequences that are derived from a commonancestor (i.e., same VH-germline gene).

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Support for this Example was provided by the Intramural Research Programof the Vaccine Research Center, National Institute of Allergy andInfectious Diseases and the National Human Genome Research Institute,National Institutes of Health, and by grants from the International AIDSVaccine Initiative's Neutralizing Antibody Consortium and by the Centerfor HIV AIDS Vaccine Immunology Grant AI 5U19 AI 067854-06 from theNational Institutes from Health. Use of sector 22 (Southeast RegionCollaborative Access team) at the Advanced Photon Source was supportedby the US Department of Energy, Basic Energy Sciences, Office ofScience, under contract number W-31-109-Eng-38. Structure factors andcoordinates for antibodies VRC03 and VRC-PG04 in complex with HIV-1gp120 have been deposited with the Protein Data Bank under accessioncodes 3SE8 and 3SE9, respectively. Applicants have also deposited deepsequencing data for donors 45 and 74 used in this Example to NCBI ShortReads Archives (SRA) under accession number SRP006992, the heavy andlight chain variable region sequences of probe-identified antibodiesVRC-PG04 and VRC-PG04b (GenBank accession numbers JN159464-JN159467),VRC-CH30, VRC-CH31 and VRC-CH32 (JN159434-JN159439), and VRC-CH33 andVRC-CH34 (JN159470-159473), as well as the sequences of genomicallyidentified neutralizers: 24 heavy chains from donor 74, 2008(JN159440-JN159463), 2 heavy chains from donor 45, 2008(JN159474-JN159475), 2 light chains from donor 45, 2001(JN159468-JN159469), and 1,561 unique sequences associated withneutralizing CDR H3 distributions with at least one low divergent member(JN157873-JN159433).

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. An isolated or non-naturally occurring humanmonoclonal antibody, wherein said monoclonal antibody neutralizes anHIV-1 virus in vitro, and further wherein the monoclonal antibody isselected from the group consisting of VRC-PG-04 or VRC-PG-05.
 2. Adiagnostic composition comprising a labeled antibody of claim 1, or afragment thereof, to detect the presence of an HIV immunogen, antigen orepitope in a sample.
 3. The composition of claim 2, wherein the sampleis a biological sample.
 4. The composition of claim 3, wherein thebiological sample is blood, semen or vaginal fluid.
 5. An isolated ornon-naturally occurring VRC-PG-04 monoclonal antibody comprising a heavychain sequence of SEQ ID NO: 6, 21 or 22 and a light chain sequence ofSEQ ID NO:
 14. 6. An isolated or non-naturally occurring variant of aVRC-PG-04 monoclonal antibody comprising a heavy chain sequence selectedfrom SEQ ID NOS: 21 or 22 and a light chain sequence selected from SEQID NOS: 33 or
 34. 7. A vector containing and expressing a nucleic acidencoding the VRC-PG-04 monoclonal antibody of claim
 5. 8. A vectorcontaining and expressing a nucleic acid encoding the VRC-PG-04monoclonal antibody of claim 6.